Transfection:Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D
Article Snippet: Rain-X (Rain-X, cat. no. 800002243) WT SC and pSC plasmids for expression in mammalian cells (for design and ordering of plasmids, see Procedure 2 ) pNeu–hMbPylRS–4xU6M15 plasmid for the expression of M. mazei pyrrolysine aminoacyl–tRNA synthetase/tRNA pair (PylRS/tRNAPyl) in mammalian cells (Addgene, cat. no. 105830) HEK293T cells (ATCC, CRL-1573; RRID: CVCL_0063) or other mammalian cell lines of interest CAUTION Cell lines should be routinely checked for mycoplasma. .. Gelatin (ThermoFisher, cat. no. 5025935) Lipofectamine 3000 (ThermoFisher, cat. no. L3000008) or other transfection reagent of choice Dulbecco’s modified Eagle medium (DMEM) (+4.5 g/ l -glucose, + l -glutamine, −sodium pyruvate) (ThermoFisher, cat. no. 11965118) Penicillin–streptomycin (ThermoFisher, cat. no. 15140122) Fetal bovine serum (FBS) (Sigma, cat. no. F0926-500ML) Hanks Buffered Salt Solution (HBSS) (Gibco, cat. no. 14025076) Trypsin–EDTA solution (ThermoFisher, cat. no. 25200072) EndoFree Plasmid DNA Maxi Kit (Omega Biotek, cat. no. NC0768143) Opti-MEM reduced serum medium (Gibco, cat. no. 31985062) Fume hood (Supreme Air, Kewaunee Scientific Corporation) Weighing balance (Mettler Toledo, cat. no. 30029077) Spatulas (Fisher Scientific, cat. no. 14373) Glassware (round-bottomed flasks, Erlenmeyer flasks, beakers, dishes) (Fisher Scientific) Buchner funnel (Fisher Scientific) Separatory funnel (Fisher Scientific) Qualitative filter paper (Cytiva, cat. no. 1001045) Magnetic stir plate (Fisher Scientific, cat. no. SP88854100) Magnetic stir bars (Fisher Scientific, cat. no. 14-513-51 Schlenk link (Thomas Scientific, cat. no. M411003) Nitrogen gas (Linde Gas & Equipment Inc., cat. no. NI K) Rotary evaporator (Büchi Rotovapor R-3 equipped with a V-700 vacuum pump, V-855 vacuum controller and a Welch 1400 DuoSeal Belt-Drive high vacuum pump) Lyophilizer (LABCONCO FreeZone 2.5 Plus with a LABCONCO rotary vane 117 vacuum pump) Mass spectrometer (ThermoFisher, Linear Trap Quadrupole Orbitrap Xcalibur 2.0 DS) NMR spectrometer (Bruker) NMR tubes (Fisher Scientific, 16800555) 50 mL conical tubes (ThermoFisher, cat. no. 12-565-271) 500 mL chromatography column 40 mm inner diameter (Fisher Scientific, cat. no. 31-500-913) Temperature-controlled shaking incubator (ThermoFisher, Model MaxQ 4000) NanoDrop spectrophotometer (ThermoFisher Model 2000C) PCR tubes (Fisher Scientific, cat. no. 14230210) Thermocyler (Bioer LifeECO) Sterile syringes, filters, needles (Fisher Scientific) Microcentrifuge tubes (BrandTech, cat. no. 780420) Shaker flasks (PYREX, cat. no. 4446500) Tabletop centrifuge (Thomas Scientific, cat. no. 75007213) Floor centrifuge (Beckman Coulter) pH meter (Fisher Scientific, cat. no. 13636XL150) Sonicator (ThermoFisher Model 505 Soni Dismembrator) CAUTION Sonicator should be in a soundproof box or ear protection should be worn. .. SDS–PAGE system (BioRad) Scanner (Azure Model 600 AZI600) Vials for LC–MS (Waters, cat. no. 186000385C) LC–MS (AB Sciex 5600 QTOF) Glass slides, 75 mm × 25 mm (Chemglass Life Sciences, cat. no. CGQ064001) Silicone rubber spacer (0.5 mm thick) (McMaster-Carr, cat. no 87315K61) 100 mm Petri dish (ThermoFisher, cat. no. FB0875712) Positive displacement pipette (Gilson, cat. no. FD10002) Zeba Spin Desalting Column (ThermoFisher, cat. no PI89882) Radiometer (series 9811–50; Cole-Parmer) UV light source (Lumen Dynamics OmniCure S1500 Spot UV Curing system with an internal 365 nm filter) Chrome photomask (PhotoSciences) Multiphoton microscope (ThorLabs Bergamo II) Standard equipment for mammalian cell culture Biological safety cabinet (Thermo Scientific, cat. no. 1385) CO 2 cell culture incubator set to 37 °C with 5% CO 2 (ThermoFisher) Water bath set to 37 °C (ThermoFisher) Centrifuge (Fisher Scientific, cat. no. 01257134) T25 flasks (Fisher Scientific, cat. no. 12565351) Hemacytometer (Hausser Scientific, cat. no. 3520) 35 mm glass-bottom dishes, 14 mm glass microwell size (Cellvis, cat. no. NC0794151) Autodesk Netfabb Premium 2023.1 ( https://www.autodesk.com ) MATLAB R2019a ( https://www.mathworks.com/products/matlab.html ) Saturated NaCl solution: dissolve 36 g of NaCl in 100 mL H 2 O Brine solution: mix 100 mL of H 2 O with 20 mL of saturated NaCl solution 4 M HCl in 1,4-dioxane: slowly add 10 mL 12 M HCl to 20 mL 1,4-dioxane in a fume hood Carbenicillin (100 mg mL −1 ): dissolve 1 g carbenicillin in 10 mL H 2 O and filter sterilize.
Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.
Article Snippet: The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger.. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity.. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide–protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly.
Modification:Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D
Article Snippet: Rain-X (Rain-X, cat. no. 800002243) WT SC and pSC plasmids for expression in mammalian cells (for design and ordering of plasmids, see Procedure 2 ) pNeu–hMbPylRS–4xU6M15 plasmid for the expression of M. mazei pyrrolysine aminoacyl–tRNA synthetase/tRNA pair (PylRS/tRNAPyl) in mammalian cells (Addgene, cat. no. 105830) HEK293T cells (ATCC, CRL-1573; RRID: CVCL_0063) or other mammalian cell lines of interest CAUTION Cell lines should be routinely checked for mycoplasma. .. Gelatin (ThermoFisher, cat. no. 5025935) Lipofectamine 3000 (ThermoFisher, cat. no. L3000008) or other transfection reagent of choice Dulbecco’s modified Eagle medium (DMEM) (+4.5 g/ l -glucose, + l -glutamine, −sodium pyruvate) (ThermoFisher, cat. no. 11965118) Penicillin–streptomycin (ThermoFisher, cat. no. 15140122) Fetal bovine serum (FBS) (Sigma, cat. no. F0926-500ML) Hanks Buffered Salt Solution (HBSS) (Gibco, cat. no. 14025076) Trypsin–EDTA solution (ThermoFisher, cat. no. 25200072) EndoFree Plasmid DNA Maxi Kit (Omega Biotek, cat. no. NC0768143) Opti-MEM reduced serum medium (Gibco, cat. no. 31985062) Fume hood (Supreme Air, Kewaunee Scientific Corporation) Weighing balance (Mettler Toledo, cat. no. 30029077) Spatulas (Fisher Scientific, cat. no. 14373) Glassware (round-bottomed flasks, Erlenmeyer flasks, beakers, dishes) (Fisher Scientific) Buchner funnel (Fisher Scientific) Separatory funnel (Fisher Scientific) Qualitative filter paper (Cytiva, cat. no. 1001045) Magnetic stir plate (Fisher Scientific, cat. no. SP88854100) Magnetic stir bars (Fisher Scientific, cat. no. 14-513-51 Schlenk link (Thomas Scientific, cat. no. M411003) Nitrogen gas (Linde Gas & Equipment Inc., cat. no. NI K) Rotary evaporator (Büchi Rotovapor R-3 equipped with a V-700 vacuum pump, V-855 vacuum controller and a Welch 1400 DuoSeal Belt-Drive high vacuum pump) Lyophilizer (LABCONCO FreeZone 2.5 Plus with a LABCONCO rotary vane 117 vacuum pump) Mass spectrometer (ThermoFisher, Linear Trap Quadrupole Orbitrap Xcalibur 2.0 DS) NMR spectrometer (Bruker) NMR tubes (Fisher Scientific, 16800555) 50 mL conical tubes (ThermoFisher, cat. no. 12-565-271) 500 mL chromatography column 40 mm inner diameter (Fisher Scientific, cat. no. 31-500-913) Temperature-controlled shaking incubator (ThermoFisher, Model MaxQ 4000) NanoDrop spectrophotometer (ThermoFisher Model 2000C) PCR tubes (Fisher Scientific, cat. no. 14230210) Thermocyler (Bioer LifeECO) Sterile syringes, filters, needles (Fisher Scientific) Microcentrifuge tubes (BrandTech, cat. no. 780420) Shaker flasks (PYREX, cat. no. 4446500) Tabletop centrifuge (Thomas Scientific, cat. no. 75007213) Floor centrifuge (Beckman Coulter) pH meter (Fisher Scientific, cat. no. 13636XL150) Sonicator (ThermoFisher Model 505 Soni Dismembrator) CAUTION Sonicator should be in a soundproof box or ear protection should be worn. .. SDS–PAGE system (BioRad) Scanner (Azure Model 600 AZI600) Vials for LC–MS (Waters, cat. no. 186000385C) LC–MS (AB Sciex 5600 QTOF) Glass slides, 75 mm × 25 mm (Chemglass Life Sciences, cat. no. CGQ064001) Silicone rubber spacer (0.5 mm thick) (McMaster-Carr, cat. no 87315K61) 100 mm Petri dish (ThermoFisher, cat. no. FB0875712) Positive displacement pipette (Gilson, cat. no. FD10002) Zeba Spin Desalting Column (ThermoFisher, cat. no PI89882) Radiometer (series 9811–50; Cole-Parmer) UV light source (Lumen Dynamics OmniCure S1500 Spot UV Curing system with an internal 365 nm filter) Chrome photomask (PhotoSciences) Multiphoton microscope (ThorLabs Bergamo II) Standard equipment for mammalian cell culture Biological safety cabinet (Thermo Scientific, cat. no. 1385) CO 2 cell culture incubator set to 37 °C with 5% CO 2 (ThermoFisher) Water bath set to 37 °C (ThermoFisher) Centrifuge (Fisher Scientific, cat. no. 01257134) T25 flasks (Fisher Scientific, cat. no. 12565351) Hemacytometer (Hausser Scientific, cat. no. 3520) 35 mm glass-bottom dishes, 14 mm glass microwell size (Cellvis, cat. no. NC0794151) Autodesk Netfabb Premium 2023.1 ( https://www.autodesk.com ) MATLAB R2019a ( https://www.mathworks.com/products/matlab.html ) Saturated NaCl solution: dissolve 36 g of NaCl in 100 mL H 2 O Brine solution: mix 100 mL of H 2 O with 20 mL of saturated NaCl solution 4 M HCl in 1,4-dioxane: slowly add 10 mL 12 M HCl to 20 mL 1,4-dioxane in a fume hood Carbenicillin (100 mg mL −1 ): dissolve 1 g carbenicillin in 10 mL H 2 O and filter sterilize.
Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.
Article Snippet: The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger.. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity.. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide–protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly.
Plasmid Preparation:Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D
Article Snippet: Rain-X (Rain-X, cat. no. 800002243) WT SC and pSC plasmids for expression in mammalian cells (for design and ordering of plasmids, see Procedure 2 ) pNeu–hMbPylRS–4xU6M15 plasmid for the expression of M. mazei pyrrolysine aminoacyl–tRNA synthetase/tRNA pair (PylRS/tRNAPyl) in mammalian cells (Addgene, cat. no. 105830) HEK293T cells (ATCC, CRL-1573; RRID: CVCL_0063) or other mammalian cell lines of interest CAUTION Cell lines should be routinely checked for mycoplasma. .. Gelatin (ThermoFisher, cat. no. 5025935) Lipofectamine 3000 (ThermoFisher, cat. no. L3000008) or other transfection reagent of choice Dulbecco’s modified Eagle medium (DMEM) (+4.5 g/ l -glucose, + l -glutamine, −sodium pyruvate) (ThermoFisher, cat. no. 11965118) Penicillin–streptomycin (ThermoFisher, cat. no. 15140122) Fetal bovine serum (FBS) (Sigma, cat. no. F0926-500ML) Hanks Buffered Salt Solution (HBSS) (Gibco, cat. no. 14025076) Trypsin–EDTA solution (ThermoFisher, cat. no. 25200072) EndoFree Plasmid DNA Maxi Kit (Omega Biotek, cat. no. NC0768143) Opti-MEM reduced serum medium (Gibco, cat. no. 31985062) Fume hood (Supreme Air, Kewaunee Scientific Corporation) Weighing balance (Mettler Toledo, cat. no. 30029077) Spatulas (Fisher Scientific, cat. no. 14373) Glassware (round-bottomed flasks, Erlenmeyer flasks, beakers, dishes) (Fisher Scientific) Buchner funnel (Fisher Scientific) Separatory funnel (Fisher Scientific) Qualitative filter paper (Cytiva, cat. no. 1001045) Magnetic stir plate (Fisher Scientific, cat. no. SP88854100) Magnetic stir bars (Fisher Scientific, cat. no. 14-513-51 Schlenk link (Thomas Scientific, cat. no. M411003) Nitrogen gas (Linde Gas & Equipment Inc., cat. no. NI K) Rotary evaporator (Büchi Rotovapor R-3 equipped with a V-700 vacuum pump, V-855 vacuum controller and a Welch 1400 DuoSeal Belt-Drive high vacuum pump) Lyophilizer (LABCONCO FreeZone 2.5 Plus with a LABCONCO rotary vane 117 vacuum pump) Mass spectrometer (ThermoFisher, Linear Trap Quadrupole Orbitrap Xcalibur 2.0 DS) NMR spectrometer (Bruker) NMR tubes (Fisher Scientific, 16800555) 50 mL conical tubes (ThermoFisher, cat. no. 12-565-271) 500 mL chromatography column 40 mm inner diameter (Fisher Scientific, cat. no. 31-500-913) Temperature-controlled shaking incubator (ThermoFisher, Model MaxQ 4000) NanoDrop spectrophotometer (ThermoFisher Model 2000C) PCR tubes (Fisher Scientific, cat. no. 14230210) Thermocyler (Bioer LifeECO) Sterile syringes, filters, needles (Fisher Scientific) Microcentrifuge tubes (BrandTech, cat. no. 780420) Shaker flasks (PYREX, cat. no. 4446500) Tabletop centrifuge (Thomas Scientific, cat. no. 75007213) Floor centrifuge (Beckman Coulter) pH meter (Fisher Scientific, cat. no. 13636XL150) Sonicator (ThermoFisher Model 505 Soni Dismembrator) CAUTION Sonicator should be in a soundproof box or ear protection should be worn. .. SDS–PAGE system (BioRad) Scanner (Azure Model 600 AZI600) Vials for LC–MS (Waters, cat. no. 186000385C) LC–MS (AB Sciex 5600 QTOF) Glass slides, 75 mm × 25 mm (Chemglass Life Sciences, cat. no. CGQ064001) Silicone rubber spacer (0.5 mm thick) (McMaster-Carr, cat. no 87315K61) 100 mm Petri dish (ThermoFisher, cat. no. FB0875712) Positive displacement pipette (Gilson, cat. no. FD10002) Zeba Spin Desalting Column (ThermoFisher, cat. no PI89882) Radiometer (series 9811–50; Cole-Parmer) UV light source (Lumen Dynamics OmniCure S1500 Spot UV Curing system with an internal 365 nm filter) Chrome photomask (PhotoSciences) Multiphoton microscope (ThorLabs Bergamo II) Standard equipment for mammalian cell culture Biological safety cabinet (Thermo Scientific, cat. no. 1385) CO 2 cell culture incubator set to 37 °C with 5% CO 2 (ThermoFisher) Water bath set to 37 °C (ThermoFisher) Centrifuge (Fisher Scientific, cat. no. 01257134) T25 flasks (Fisher Scientific, cat. no. 12565351) Hemacytometer (Hausser Scientific, cat. no. 3520) 35 mm glass-bottom dishes, 14 mm glass microwell size (Cellvis, cat. no. NC0794151) Autodesk Netfabb Premium 2023.1 ( https://www.autodesk.com ) MATLAB R2019a ( https://www.mathworks.com/products/matlab.html ) Saturated NaCl solution: dissolve 36 g of NaCl in 100 mL H 2 O Brine solution: mix 100 mL of H 2 O with 20 mL of saturated NaCl solution 4 M HCl in 1,4-dioxane: slowly add 10 mL 12 M HCl to 20 mL 1,4-dioxane in a fume hood Carbenicillin (100 mg mL −1 ): dissolve 1 g carbenicillin in 10 mL H 2 O and filter sterilize.
Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.
Article Snippet: The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger.. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity.. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide–protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly.
Hood:Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D
Article Snippet: Rain-X (Rain-X, cat. no. 800002243) WT SC and pSC plasmids for expression in mammalian cells (for design and ordering of plasmids, see Procedure 2 ) pNeu–hMbPylRS–4xU6M15 plasmid for the expression of M. mazei pyrrolysine aminoacyl–tRNA synthetase/tRNA pair (PylRS/tRNAPyl) in mammalian cells (Addgene, cat. no. 105830) HEK293T cells (ATCC, CRL-1573; RRID: CVCL_0063) or other mammalian cell lines of interest CAUTION Cell lines should be routinely checked for mycoplasma. .. Gelatin (ThermoFisher, cat. no. 5025935) Lipofectamine 3000 (ThermoFisher, cat. no. L3000008) or other transfection reagent of choice Dulbecco’s modified Eagle medium (DMEM) (+4.5 g/ l -glucose, + l -glutamine, −sodium pyruvate) (ThermoFisher, cat. no. 11965118) Penicillin–streptomycin (ThermoFisher, cat. no. 15140122) Fetal bovine serum (FBS) (Sigma, cat. no. F0926-500ML) Hanks Buffered Salt Solution (HBSS) (Gibco, cat. no. 14025076) Trypsin–EDTA solution (ThermoFisher, cat. no. 25200072) EndoFree Plasmid DNA Maxi Kit (Omega Biotek, cat. no. NC0768143) Opti-MEM reduced serum medium (Gibco, cat. no. 31985062) Fume hood (Supreme Air, Kewaunee Scientific Corporation) Weighing balance (Mettler Toledo, cat. no. 30029077) Spatulas (Fisher Scientific, cat. no. 14373) Glassware (round-bottomed flasks, Erlenmeyer flasks, beakers, dishes) (Fisher Scientific) Buchner funnel (Fisher Scientific) Separatory funnel (Fisher Scientific) Qualitative filter paper (Cytiva, cat. no. 1001045) Magnetic stir plate (Fisher Scientific, cat. no. SP88854100) Magnetic stir bars (Fisher Scientific, cat. no. 14-513-51 Schlenk link (Thomas Scientific, cat. no. M411003) Nitrogen gas (Linde Gas & Equipment Inc., cat. no. NI K) Rotary evaporator (Büchi Rotovapor R-3 equipped with a V-700 vacuum pump, V-855 vacuum controller and a Welch 1400 DuoSeal Belt-Drive high vacuum pump) Lyophilizer (LABCONCO FreeZone 2.5 Plus with a LABCONCO rotary vane 117 vacuum pump) Mass spectrometer (ThermoFisher, Linear Trap Quadrupole Orbitrap Xcalibur 2.0 DS) NMR spectrometer (Bruker) NMR tubes (Fisher Scientific, 16800555) 50 mL conical tubes (ThermoFisher, cat. no. 12-565-271) 500 mL chromatography column 40 mm inner diameter (Fisher Scientific, cat. no. 31-500-913) Temperature-controlled shaking incubator (ThermoFisher, Model MaxQ 4000) NanoDrop spectrophotometer (ThermoFisher Model 2000C) PCR tubes (Fisher Scientific, cat. no. 14230210) Thermocyler (Bioer LifeECO) Sterile syringes, filters, needles (Fisher Scientific) Microcentrifuge tubes (BrandTech, cat. no. 780420) Shaker flasks (PYREX, cat. no. 4446500) Tabletop centrifuge (Thomas Scientific, cat. no. 75007213) Floor centrifuge (Beckman Coulter) pH meter (Fisher Scientific, cat. no. 13636XL150) Sonicator (ThermoFisher Model 505 Soni Dismembrator) CAUTION Sonicator should be in a soundproof box or ear protection should be worn. .. SDS–PAGE system (BioRad) Scanner (Azure Model 600 AZI600) Vials for LC–MS (Waters, cat. no. 186000385C) LC–MS (AB Sciex 5600 QTOF) Glass slides, 75 mm × 25 mm (Chemglass Life Sciences, cat. no. CGQ064001) Silicone rubber spacer (0.5 mm thick) (McMaster-Carr, cat. no 87315K61) 100 mm Petri dish (ThermoFisher, cat. no. FB0875712) Positive displacement pipette (Gilson, cat. no. FD10002) Zeba Spin Desalting Column (ThermoFisher, cat. no PI89882) Radiometer (series 9811–50; Cole-Parmer) UV light source (Lumen Dynamics OmniCure S1500 Spot UV Curing system with an internal 365 nm filter) Chrome photomask (PhotoSciences) Multiphoton microscope (ThorLabs Bergamo II) Standard equipment for mammalian cell culture Biological safety cabinet (Thermo Scientific, cat. no. 1385) CO 2 cell culture incubator set to 37 °C with 5% CO 2 (ThermoFisher) Water bath set to 37 °C (ThermoFisher) Centrifuge (Fisher Scientific, cat. no. 01257134) T25 flasks (Fisher Scientific, cat. no. 12565351) Hemacytometer (Hausser Scientific, cat. no. 3520) 35 mm glass-bottom dishes, 14 mm glass microwell size (Cellvis, cat. no. NC0794151) Autodesk Netfabb Premium 2023.1 ( https://www.autodesk.com ) MATLAB R2019a ( https://www.mathworks.com/products/matlab.html ) Saturated NaCl solution: dissolve 36 g of NaCl in 100 mL H 2 O Brine solution: mix 100 mL of H 2 O with 20 mL of saturated NaCl solution 4 M HCl in 1,4-dioxane: slowly add 10 mL 12 M HCl to 20 mL 1,4-dioxane in a fume hood Carbenicillin (100 mg mL −1 ): dissolve 1 g carbenicillin in 10 mL H 2 O and filter sterilize.
Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.
Article Snippet: The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger.. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity.. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide–protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly.
Mass Spectrometry:Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D
Article Snippet: Rain-X (Rain-X, cat. no. 800002243) WT SC and pSC plasmids for expression in mammalian cells (for design and ordering of plasmids, see Procedure 2 ) pNeu–hMbPylRS–4xU6M15 plasmid for the expression of M. mazei pyrrolysine aminoacyl–tRNA synthetase/tRNA pair (PylRS/tRNAPyl) in mammalian cells (Addgene, cat. no. 105830) HEK293T cells (ATCC, CRL-1573; RRID: CVCL_0063) or other mammalian cell lines of interest CAUTION Cell lines should be routinely checked for mycoplasma. .. Gelatin (ThermoFisher, cat. no. 5025935) Lipofectamine 3000 (ThermoFisher, cat. no. L3000008) or other transfection reagent of choice Dulbecco’s modified Eagle medium (DMEM) (+4.5 g/ l -glucose, + l -glutamine, −sodium pyruvate) (ThermoFisher, cat. no. 11965118) Penicillin–streptomycin (ThermoFisher, cat. no. 15140122) Fetal bovine serum (FBS) (Sigma, cat. no. F0926-500ML) Hanks Buffered Salt Solution (HBSS) (Gibco, cat. no. 14025076) Trypsin–EDTA solution (ThermoFisher, cat. no. 25200072) EndoFree Plasmid DNA Maxi Kit (Omega Biotek, cat. no. NC0768143) Opti-MEM reduced serum medium (Gibco, cat. no. 31985062) Fume hood (Supreme Air, Kewaunee Scientific Corporation) Weighing balance (Mettler Toledo, cat. no. 30029077) Spatulas (Fisher Scientific, cat. no. 14373) Glassware (round-bottomed flasks, Erlenmeyer flasks, beakers, dishes) (Fisher Scientific) Buchner funnel (Fisher Scientific) Separatory funnel (Fisher Scientific) Qualitative filter paper (Cytiva, cat. no. 1001045) Magnetic stir plate (Fisher Scientific, cat. no. SP88854100) Magnetic stir bars (Fisher Scientific, cat. no. 14-513-51 Schlenk link (Thomas Scientific, cat. no. M411003) Nitrogen gas (Linde Gas & Equipment Inc., cat. no. NI K) Rotary evaporator (Büchi Rotovapor R-3 equipped with a V-700 vacuum pump, V-855 vacuum controller and a Welch 1400 DuoSeal Belt-Drive high vacuum pump) Lyophilizer (LABCONCO FreeZone 2.5 Plus with a LABCONCO rotary vane 117 vacuum pump) Mass spectrometer (ThermoFisher, Linear Trap Quadrupole Orbitrap Xcalibur 2.0 DS) NMR spectrometer (Bruker) NMR tubes (Fisher Scientific, 16800555) 50 mL conical tubes (ThermoFisher, cat. no. 12-565-271) 500 mL chromatography column 40 mm inner diameter (Fisher Scientific, cat. no. 31-500-913) Temperature-controlled shaking incubator (ThermoFisher, Model MaxQ 4000) NanoDrop spectrophotometer (ThermoFisher Model 2000C) PCR tubes (Fisher Scientific, cat. no. 14230210) Thermocyler (Bioer LifeECO) Sterile syringes, filters, needles (Fisher Scientific) Microcentrifuge tubes (BrandTech, cat. no. 780420) Shaker flasks (PYREX, cat. no. 4446500) Tabletop centrifuge (Thomas Scientific, cat. no. 75007213) Floor centrifuge (Beckman Coulter) pH meter (Fisher Scientific, cat. no. 13636XL150) Sonicator (ThermoFisher Model 505 Soni Dismembrator) CAUTION Sonicator should be in a soundproof box or ear protection should be worn. .. SDS–PAGE system (BioRad) Scanner (Azure Model 600 AZI600) Vials for LC–MS (Waters, cat. no. 186000385C) LC–MS (AB Sciex 5600 QTOF) Glass slides, 75 mm × 25 mm (Chemglass Life Sciences, cat. no. CGQ064001) Silicone rubber spacer (0.5 mm thick) (McMaster-Carr, cat. no 87315K61) 100 mm Petri dish (ThermoFisher, cat. no. FB0875712) Positive displacement pipette (Gilson, cat. no. FD10002) Zeba Spin Desalting Column (ThermoFisher, cat. no PI89882) Radiometer (series 9811–50; Cole-Parmer) UV light source (Lumen Dynamics OmniCure S1500 Spot UV Curing system with an internal 365 nm filter) Chrome photomask (PhotoSciences) Multiphoton microscope (ThorLabs Bergamo II) Standard equipment for mammalian cell culture Biological safety cabinet (Thermo Scientific, cat. no. 1385) CO 2 cell culture incubator set to 37 °C with 5% CO 2 (ThermoFisher) Water bath set to 37 °C (ThermoFisher) Centrifuge (Fisher Scientific, cat. no. 01257134) T25 flasks (Fisher Scientific, cat. no. 12565351) Hemacytometer (Hausser Scientific, cat. no. 3520) 35 mm glass-bottom dishes, 14 mm glass microwell size (Cellvis, cat. no. NC0794151) Autodesk Netfabb Premium 2023.1 ( https://www.autodesk.com ) MATLAB R2019a ( https://www.mathworks.com/products/matlab.html ) Saturated NaCl solution: dissolve 36 g of NaCl in 100 mL H 2 O Brine solution: mix 100 mL of H 2 O with 20 mL of saturated NaCl solution 4 M HCl in 1,4-dioxane: slowly add 10 mL 12 M HCl to 20 mL 1,4-dioxane in a fume hood Carbenicillin (100 mg mL −1 ): dissolve 1 g carbenicillin in 10 mL H 2 O and filter sterilize.
Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.
Article Snippet: The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger.. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity.. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide–protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly.
Nuclear Magnetic Resonance:Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D
Article Snippet: Rain-X (Rain-X, cat. no. 800002243) WT SC and pSC plasmids for expression in mammalian cells (for design and ordering of plasmids, see Procedure 2 ) pNeu–hMbPylRS–4xU6M15 plasmid for the expression of M. mazei pyrrolysine aminoacyl–tRNA synthetase/tRNA pair (PylRS/tRNAPyl) in mammalian cells (Addgene, cat. no. 105830) HEK293T cells (ATCC, CRL-1573; RRID: CVCL_0063) or other mammalian cell lines of interest CAUTION Cell lines should be routinely checked for mycoplasma. .. Gelatin (ThermoFisher, cat. no. 5025935) Lipofectamine 3000 (ThermoFisher, cat. no. L3000008) or other transfection reagent of choice Dulbecco’s modified Eagle medium (DMEM) (+4.5 g/ l -glucose, + l -glutamine, −sodium pyruvate) (ThermoFisher, cat. no. 11965118) Penicillin–streptomycin (ThermoFisher, cat. no. 15140122) Fetal bovine serum (FBS) (Sigma, cat. no. F0926-500ML) Hanks Buffered Salt Solution (HBSS) (Gibco, cat. no. 14025076) Trypsin–EDTA solution (ThermoFisher, cat. no. 25200072) EndoFree Plasmid DNA Maxi Kit (Omega Biotek, cat. no. NC0768143) Opti-MEM reduced serum medium (Gibco, cat. no. 31985062) Fume hood (Supreme Air, Kewaunee Scientific Corporation) Weighing balance (Mettler Toledo, cat. no. 30029077) Spatulas (Fisher Scientific, cat. no. 14373) Glassware (round-bottomed flasks, Erlenmeyer flasks, beakers, dishes) (Fisher Scientific) Buchner funnel (Fisher Scientific) Separatory funnel (Fisher Scientific) Qualitative filter paper (Cytiva, cat. no. 1001045) Magnetic stir plate (Fisher Scientific, cat. no. SP88854100) Magnetic stir bars (Fisher Scientific, cat. no. 14-513-51 Schlenk link (Thomas Scientific, cat. no. M411003) Nitrogen gas (Linde Gas & Equipment Inc., cat. no. NI K) Rotary evaporator (Büchi Rotovapor R-3 equipped with a V-700 vacuum pump, V-855 vacuum controller and a Welch 1400 DuoSeal Belt-Drive high vacuum pump) Lyophilizer (LABCONCO FreeZone 2.5 Plus with a LABCONCO rotary vane 117 vacuum pump) Mass spectrometer (ThermoFisher, Linear Trap Quadrupole Orbitrap Xcalibur 2.0 DS) NMR spectrometer (Bruker) NMR tubes (Fisher Scientific, 16800555) 50 mL conical tubes (ThermoFisher, cat. no. 12-565-271) 500 mL chromatography column 40 mm inner diameter (Fisher Scientific, cat. no. 31-500-913) Temperature-controlled shaking incubator (ThermoFisher, Model MaxQ 4000) NanoDrop spectrophotometer (ThermoFisher Model 2000C) PCR tubes (Fisher Scientific, cat. no. 14230210) Thermocyler (Bioer LifeECO) Sterile syringes, filters, needles (Fisher Scientific) Microcentrifuge tubes (BrandTech, cat. no. 780420) Shaker flasks (PYREX, cat. no. 4446500) Tabletop centrifuge (Thomas Scientific, cat. no. 75007213) Floor centrifuge (Beckman Coulter) pH meter (Fisher Scientific, cat. no. 13636XL150) Sonicator (ThermoFisher Model 505 Soni Dismembrator) CAUTION Sonicator should be in a soundproof box or ear protection should be worn. .. SDS–PAGE system (BioRad) Scanner (Azure Model 600 AZI600) Vials for LC–MS (Waters, cat. no. 186000385C) LC–MS (AB Sciex 5600 QTOF) Glass slides, 75 mm × 25 mm (Chemglass Life Sciences, cat. no. CGQ064001) Silicone rubber spacer (0.5 mm thick) (McMaster-Carr, cat. no 87315K61) 100 mm Petri dish (ThermoFisher, cat. no. FB0875712) Positive displacement pipette (Gilson, cat. no. FD10002) Zeba Spin Desalting Column (ThermoFisher, cat. no PI89882) Radiometer (series 9811–50; Cole-Parmer) UV light source (Lumen Dynamics OmniCure S1500 Spot UV Curing system with an internal 365 nm filter) Chrome photomask (PhotoSciences) Multiphoton microscope (ThorLabs Bergamo II) Standard equipment for mammalian cell culture Biological safety cabinet (Thermo Scientific, cat. no. 1385) CO 2 cell culture incubator set to 37 °C with 5% CO 2 (ThermoFisher) Water bath set to 37 °C (ThermoFisher) Centrifuge (Fisher Scientific, cat. no. 01257134) T25 flasks (Fisher Scientific, cat. no. 12565351) Hemacytometer (Hausser Scientific, cat. no. 3520) 35 mm glass-bottom dishes, 14 mm glass microwell size (Cellvis, cat. no. NC0794151) Autodesk Netfabb Premium 2023.1 ( https://www.autodesk.com ) MATLAB R2019a ( https://www.mathworks.com/products/matlab.html ) Saturated NaCl solution: dissolve 36 g of NaCl in 100 mL H 2 O Brine solution: mix 100 mL of H 2 O with 20 mL of saturated NaCl solution 4 M HCl in 1,4-dioxane: slowly add 10 mL 12 M HCl to 20 mL 1,4-dioxane in a fume hood Carbenicillin (100 mg mL −1 ): dissolve 1 g carbenicillin in 10 mL H 2 O and filter sterilize.
Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.
Article Snippet: The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger.. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity.. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide–protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly.
Chromatography:Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D
Article Snippet: Rain-X (Rain-X, cat. no. 800002243) WT SC and pSC plasmids for expression in mammalian cells (for design and ordering of plasmids, see Procedure 2 ) pNeu–hMbPylRS–4xU6M15 plasmid for the expression of M. mazei pyrrolysine aminoacyl–tRNA synthetase/tRNA pair (PylRS/tRNAPyl) in mammalian cells (Addgene, cat. no. 105830) HEK293T cells (ATCC, CRL-1573; RRID: CVCL_0063) or other mammalian cell lines of interest CAUTION Cell lines should be routinely checked for mycoplasma. .. Gelatin (ThermoFisher, cat. no. 5025935) Lipofectamine 3000 (ThermoFisher, cat. no. L3000008) or other transfection reagent of choice Dulbecco’s modified Eagle medium (DMEM) (+4.5 g/ l -glucose, + l -glutamine, −sodium pyruvate) (ThermoFisher, cat. no. 11965118) Penicillin–streptomycin (ThermoFisher, cat. no. 15140122) Fetal bovine serum (FBS) (Sigma, cat. no. F0926-500ML) Hanks Buffered Salt Solution (HBSS) (Gibco, cat. no. 14025076) Trypsin–EDTA solution (ThermoFisher, cat. no. 25200072) EndoFree Plasmid DNA Maxi Kit (Omega Biotek, cat. no. NC0768143) Opti-MEM reduced serum medium (Gibco, cat. no. 31985062) Fume hood (Supreme Air, Kewaunee Scientific Corporation) Weighing balance (Mettler Toledo, cat. no. 30029077) Spatulas (Fisher Scientific, cat. no. 14373) Glassware (round-bottomed flasks, Erlenmeyer flasks, beakers, dishes) (Fisher Scientific) Buchner funnel (Fisher Scientific) Separatory funnel (Fisher Scientific) Qualitative filter paper (Cytiva, cat. no. 1001045) Magnetic stir plate (Fisher Scientific, cat. no. SP88854100) Magnetic stir bars (Fisher Scientific, cat. no. 14-513-51 Schlenk link (Thomas Scientific, cat. no. M411003) Nitrogen gas (Linde Gas & Equipment Inc., cat. no. NI K) Rotary evaporator (Büchi Rotovapor R-3 equipped with a V-700 vacuum pump, V-855 vacuum controller and a Welch 1400 DuoSeal Belt-Drive high vacuum pump) Lyophilizer (LABCONCO FreeZone 2.5 Plus with a LABCONCO rotary vane 117 vacuum pump) Mass spectrometer (ThermoFisher, Linear Trap Quadrupole Orbitrap Xcalibur 2.0 DS) NMR spectrometer (Bruker) NMR tubes (Fisher Scientific, 16800555) 50 mL conical tubes (ThermoFisher, cat. no. 12-565-271) 500 mL chromatography column 40 mm inner diameter (Fisher Scientific, cat. no. 31-500-913) Temperature-controlled shaking incubator (ThermoFisher, Model MaxQ 4000) NanoDrop spectrophotometer (ThermoFisher Model 2000C) PCR tubes (Fisher Scientific, cat. no. 14230210) Thermocyler (Bioer LifeECO) Sterile syringes, filters, needles (Fisher Scientific) Microcentrifuge tubes (BrandTech, cat. no. 780420) Shaker flasks (PYREX, cat. no. 4446500) Tabletop centrifuge (Thomas Scientific, cat. no. 75007213) Floor centrifuge (Beckman Coulter) pH meter (Fisher Scientific, cat. no. 13636XL150) Sonicator (ThermoFisher Model 505 Soni Dismembrator) CAUTION Sonicator should be in a soundproof box or ear protection should be worn. .. SDS–PAGE system (BioRad) Scanner (Azure Model 600 AZI600) Vials for LC–MS (Waters, cat. no. 186000385C) LC–MS (AB Sciex 5600 QTOF) Glass slides, 75 mm × 25 mm (Chemglass Life Sciences, cat. no. CGQ064001) Silicone rubber spacer (0.5 mm thick) (McMaster-Carr, cat. no 87315K61) 100 mm Petri dish (ThermoFisher, cat. no. FB0875712) Positive displacement pipette (Gilson, cat. no. FD10002) Zeba Spin Desalting Column (ThermoFisher, cat. no PI89882) Radiometer (series 9811–50; Cole-Parmer) UV light source (Lumen Dynamics OmniCure S1500 Spot UV Curing system with an internal 365 nm filter) Chrome photomask (PhotoSciences) Multiphoton microscope (ThorLabs Bergamo II) Standard equipment for mammalian cell culture Biological safety cabinet (Thermo Scientific, cat. no. 1385) CO 2 cell culture incubator set to 37 °C with 5% CO 2 (ThermoFisher) Water bath set to 37 °C (ThermoFisher) Centrifuge (Fisher Scientific, cat. no. 01257134) T25 flasks (Fisher Scientific, cat. no. 12565351) Hemacytometer (Hausser Scientific, cat. no. 3520) 35 mm glass-bottom dishes, 14 mm glass microwell size (Cellvis, cat. no. NC0794151) Autodesk Netfabb Premium 2023.1 ( https://www.autodesk.com ) MATLAB R2019a ( https://www.mathworks.com/products/matlab.html ) Saturated NaCl solution: dissolve 36 g of NaCl in 100 mL H 2 O Brine solution: mix 100 mL of H 2 O with 20 mL of saturated NaCl solution 4 M HCl in 1,4-dioxane: slowly add 10 mL 12 M HCl to 20 mL 1,4-dioxane in a fume hood Carbenicillin (100 mg mL −1 ): dissolve 1 g carbenicillin in 10 mL H 2 O and filter sterilize.
Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.
Article Snippet: The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger.. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity.. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide–protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly.
Spectrophotometry:Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D
Article Snippet: Rain-X (Rain-X, cat. no. 800002243) WT SC and pSC plasmids for expression in mammalian cells (for design and ordering of plasmids, see Procedure 2 ) pNeu–hMbPylRS–4xU6M15 plasmid for the expression of M. mazei pyrrolysine aminoacyl–tRNA synthetase/tRNA pair (PylRS/tRNAPyl) in mammalian cells (Addgene, cat. no. 105830) HEK293T cells (ATCC, CRL-1573; RRID: CVCL_0063) or other mammalian cell lines of interest CAUTION Cell lines should be routinely checked for mycoplasma. .. Gelatin (ThermoFisher, cat. no. 5025935) Lipofectamine 3000 (ThermoFisher, cat. no. L3000008) or other transfection reagent of choice Dulbecco’s modified Eagle medium (DMEM) (+4.5 g/ l -glucose, + l -glutamine, −sodium pyruvate) (ThermoFisher, cat. no. 11965118) Penicillin–streptomycin (ThermoFisher, cat. no. 15140122) Fetal bovine serum (FBS) (Sigma, cat. no. F0926-500ML) Hanks Buffered Salt Solution (HBSS) (Gibco, cat. no. 14025076) Trypsin–EDTA solution (ThermoFisher, cat. no. 25200072) EndoFree Plasmid DNA Maxi Kit (Omega Biotek, cat. no. NC0768143) Opti-MEM reduced serum medium (Gibco, cat. no. 31985062) Fume hood (Supreme Air, Kewaunee Scientific Corporation) Weighing balance (Mettler Toledo, cat. no. 30029077) Spatulas (Fisher Scientific, cat. no. 14373) Glassware (round-bottomed flasks, Erlenmeyer flasks, beakers, dishes) (Fisher Scientific) Buchner funnel (Fisher Scientific) Separatory funnel (Fisher Scientific) Qualitative filter paper (Cytiva, cat. no. 1001045) Magnetic stir plate (Fisher Scientific, cat. no. SP88854100) Magnetic stir bars (Fisher Scientific, cat. no. 14-513-51 Schlenk link (Thomas Scientific, cat. no. M411003) Nitrogen gas (Linde Gas & Equipment Inc., cat. no. NI K) Rotary evaporator (Büchi Rotovapor R-3 equipped with a V-700 vacuum pump, V-855 vacuum controller and a Welch 1400 DuoSeal Belt-Drive high vacuum pump) Lyophilizer (LABCONCO FreeZone 2.5 Plus with a LABCONCO rotary vane 117 vacuum pump) Mass spectrometer (ThermoFisher, Linear Trap Quadrupole Orbitrap Xcalibur 2.0 DS) NMR spectrometer (Bruker) NMR tubes (Fisher Scientific, 16800555) 50 mL conical tubes (ThermoFisher, cat. no. 12-565-271) 500 mL chromatography column 40 mm inner diameter (Fisher Scientific, cat. no. 31-500-913) Temperature-controlled shaking incubator (ThermoFisher, Model MaxQ 4000) NanoDrop spectrophotometer (ThermoFisher Model 2000C) PCR tubes (Fisher Scientific, cat. no. 14230210) Thermocyler (Bioer LifeECO) Sterile syringes, filters, needles (Fisher Scientific) Microcentrifuge tubes (BrandTech, cat. no. 780420) Shaker flasks (PYREX, cat. no. 4446500) Tabletop centrifuge (Thomas Scientific, cat. no. 75007213) Floor centrifuge (Beckman Coulter) pH meter (Fisher Scientific, cat. no. 13636XL150) Sonicator (ThermoFisher Model 505 Soni Dismembrator) CAUTION Sonicator should be in a soundproof box or ear protection should be worn. .. SDS–PAGE system (BioRad) Scanner (Azure Model 600 AZI600) Vials for LC–MS (Waters, cat. no. 186000385C) LC–MS (AB Sciex 5600 QTOF) Glass slides, 75 mm × 25 mm (Chemglass Life Sciences, cat. no. CGQ064001) Silicone rubber spacer (0.5 mm thick) (McMaster-Carr, cat. no 87315K61) 100 mm Petri dish (ThermoFisher, cat. no. FB0875712) Positive displacement pipette (Gilson, cat. no. FD10002) Zeba Spin Desalting Column (ThermoFisher, cat. no PI89882) Radiometer (series 9811–50; Cole-Parmer) UV light source (Lumen Dynamics OmniCure S1500 Spot UV Curing system with an internal 365 nm filter) Chrome photomask (PhotoSciences) Multiphoton microscope (ThorLabs Bergamo II) Standard equipment for mammalian cell culture Biological safety cabinet (Thermo Scientific, cat. no. 1385) CO 2 cell culture incubator set to 37 °C with 5% CO 2 (ThermoFisher) Water bath set to 37 °C (ThermoFisher) Centrifuge (Fisher Scientific, cat. no. 01257134) T25 flasks (Fisher Scientific, cat. no. 12565351) Hemacytometer (Hausser Scientific, cat. no. 3520) 35 mm glass-bottom dishes, 14 mm glass microwell size (Cellvis, cat. no. NC0794151) Autodesk Netfabb Premium 2023.1 ( https://www.autodesk.com ) MATLAB R2019a ( https://www.mathworks.com/products/matlab.html ) Saturated NaCl solution: dissolve 36 g of NaCl in 100 mL H 2 O Brine solution: mix 100 mL of H 2 O with 20 mL of saturated NaCl solution 4 M HCl in 1,4-dioxane: slowly add 10 mL 12 M HCl to 20 mL 1,4-dioxane in a fume hood Carbenicillin (100 mg mL −1 ): dissolve 1 g carbenicillin in 10 mL H 2 O and filter sterilize.
Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.
Article Snippet: The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger.. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity.. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide–protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly.
Polymerase Chain Reaction:Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D
Article Snippet: Rain-X (Rain-X, cat. no. 800002243) WT SC and pSC plasmids for expression in mammalian cells (for design and ordering of plasmids, see Procedure 2 ) pNeu–hMbPylRS–4xU6M15 plasmid for the expression of M. mazei pyrrolysine aminoacyl–tRNA synthetase/tRNA pair (PylRS/tRNAPyl) in mammalian cells (Addgene, cat. no. 105830) HEK293T cells (ATCC, CRL-1573; RRID: CVCL_0063) or other mammalian cell lines of interest CAUTION Cell lines should be routinely checked for mycoplasma. .. Gelatin (ThermoFisher, cat. no. 5025935) Lipofectamine 3000 (ThermoFisher, cat. no. L3000008) or other transfection reagent of choice Dulbecco’s modified Eagle medium (DMEM) (+4.5 g/ l -glucose, + l -glutamine, −sodium pyruvate) (ThermoFisher, cat. no. 11965118) Penicillin–streptomycin (ThermoFisher, cat. no. 15140122) Fetal bovine serum (FBS) (Sigma, cat. no. F0926-500ML) Hanks Buffered Salt Solution (HBSS) (Gibco, cat. no. 14025076) Trypsin–EDTA solution (ThermoFisher, cat. no. 25200072) EndoFree Plasmid DNA Maxi Kit (Omega Biotek, cat. no. NC0768143) Opti-MEM reduced serum medium (Gibco, cat. no. 31985062) Fume hood (Supreme Air, Kewaunee Scientific Corporation) Weighing balance (Mettler Toledo, cat. no. 30029077) Spatulas (Fisher Scientific, cat. no. 14373) Glassware (round-bottomed flasks, Erlenmeyer flasks, beakers, dishes) (Fisher Scientific) Buchner funnel (Fisher Scientific) Separatory funnel (Fisher Scientific) Qualitative filter paper (Cytiva, cat. no. 1001045) Magnetic stir plate (Fisher Scientific, cat. no. SP88854100) Magnetic stir bars (Fisher Scientific, cat. no. 14-513-51 Schlenk link (Thomas Scientific, cat. no. M411003) Nitrogen gas (Linde Gas & Equipment Inc., cat. no. NI K) Rotary evaporator (Büchi Rotovapor R-3 equipped with a V-700 vacuum pump, V-855 vacuum controller and a Welch 1400 DuoSeal Belt-Drive high vacuum pump) Lyophilizer (LABCONCO FreeZone 2.5 Plus with a LABCONCO rotary vane 117 vacuum pump) Mass spectrometer (ThermoFisher, Linear Trap Quadrupole Orbitrap Xcalibur 2.0 DS) NMR spectrometer (Bruker) NMR tubes (Fisher Scientific, 16800555) 50 mL conical tubes (ThermoFisher, cat. no. 12-565-271) 500 mL chromatography column 40 mm inner diameter (Fisher Scientific, cat. no. 31-500-913) Temperature-controlled shaking incubator (ThermoFisher, Model MaxQ 4000) NanoDrop spectrophotometer (ThermoFisher Model 2000C) PCR tubes (Fisher Scientific, cat. no. 14230210) Thermocyler (Bioer LifeECO) Sterile syringes, filters, needles (Fisher Scientific) Microcentrifuge tubes (BrandTech, cat. no. 780420) Shaker flasks (PYREX, cat. no. 4446500) Tabletop centrifuge (Thomas Scientific, cat. no. 75007213) Floor centrifuge (Beckman Coulter) pH meter (Fisher Scientific, cat. no. 13636XL150) Sonicator (ThermoFisher Model 505 Soni Dismembrator) CAUTION Sonicator should be in a soundproof box or ear protection should be worn. .. SDS–PAGE system (BioRad) Scanner (Azure Model 600 AZI600) Vials for LC–MS (Waters, cat. no. 186000385C) LC–MS (AB Sciex 5600 QTOF) Glass slides, 75 mm × 25 mm (Chemglass Life Sciences, cat. no. CGQ064001) Silicone rubber spacer (0.5 mm thick) (McMaster-Carr, cat. no 87315K61) 100 mm Petri dish (ThermoFisher, cat. no. FB0875712) Positive displacement pipette (Gilson, cat. no. FD10002) Zeba Spin Desalting Column (ThermoFisher, cat. no PI89882) Radiometer (series 9811–50; Cole-Parmer) UV light source (Lumen Dynamics OmniCure S1500 Spot UV Curing system with an internal 365 nm filter) Chrome photomask (PhotoSciences) Multiphoton microscope (ThorLabs Bergamo II) Standard equipment for mammalian cell culture Biological safety cabinet (Thermo Scientific, cat. no. 1385) CO 2 cell culture incubator set to 37 °C with 5% CO 2 (ThermoFisher) Water bath set to 37 °C (ThermoFisher) Centrifuge (Fisher Scientific, cat. no. 01257134) T25 flasks (Fisher Scientific, cat. no. 12565351) Hemacytometer (Hausser Scientific, cat. no. 3520) 35 mm glass-bottom dishes, 14 mm glass microwell size (Cellvis, cat. no. NC0794151) Autodesk Netfabb Premium 2023.1 ( https://www.autodesk.com ) MATLAB R2019a ( https://www.mathworks.com/products/matlab.html ) Saturated NaCl solution: dissolve 36 g of NaCl in 100 mL H 2 O Brine solution: mix 100 mL of H 2 O with 20 mL of saturated NaCl solution 4 M HCl in 1,4-dioxane: slowly add 10 mL 12 M HCl to 20 mL 1,4-dioxane in a fume hood Carbenicillin (100 mg mL −1 ): dissolve 1 g carbenicillin in 10 mL H 2 O and filter sterilize.
Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.
Article Snippet: The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger.. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity.. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide–protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly.
Sterility:Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D
Article Snippet: Rain-X (Rain-X, cat. no. 800002243) WT SC and pSC plasmids for expression in mammalian cells (for design and ordering of plasmids, see Procedure 2 ) pNeu–hMbPylRS–4xU6M15 plasmid for the expression of M. mazei pyrrolysine aminoacyl–tRNA synthetase/tRNA pair (PylRS/tRNAPyl) in mammalian cells (Addgene, cat. no. 105830) HEK293T cells (ATCC, CRL-1573; RRID: CVCL_0063) or other mammalian cell lines of interest CAUTION Cell lines should be routinely checked for mycoplasma. .. Gelatin (ThermoFisher, cat. no. 5025935) Lipofectamine 3000 (ThermoFisher, cat. no. L3000008) or other transfection reagent of choice Dulbecco’s modified Eagle medium (DMEM) (+4.5 g/ l -glucose, + l -glutamine, −sodium pyruvate) (ThermoFisher, cat. no. 11965118) Penicillin–streptomycin (ThermoFisher, cat. no. 15140122) Fetal bovine serum (FBS) (Sigma, cat. no. F0926-500ML) Hanks Buffered Salt Solution (HBSS) (Gibco, cat. no. 14025076) Trypsin–EDTA solution (ThermoFisher, cat. no. 25200072) EndoFree Plasmid DNA Maxi Kit (Omega Biotek, cat. no. NC0768143) Opti-MEM reduced serum medium (Gibco, cat. no. 31985062) Fume hood (Supreme Air, Kewaunee Scientific Corporation) Weighing balance (Mettler Toledo, cat. no. 30029077) Spatulas (Fisher Scientific, cat. no. 14373) Glassware (round-bottomed flasks, Erlenmeyer flasks, beakers, dishes) (Fisher Scientific) Buchner funnel (Fisher Scientific) Separatory funnel (Fisher Scientific) Qualitative filter paper (Cytiva, cat. no. 1001045) Magnetic stir plate (Fisher Scientific, cat. no. SP88854100) Magnetic stir bars (Fisher Scientific, cat. no. 14-513-51 Schlenk link (Thomas Scientific, cat. no. M411003) Nitrogen gas (Linde Gas & Equipment Inc., cat. no. NI K) Rotary evaporator (Büchi Rotovapor R-3 equipped with a V-700 vacuum pump, V-855 vacuum controller and a Welch 1400 DuoSeal Belt-Drive high vacuum pump) Lyophilizer (LABCONCO FreeZone 2.5 Plus with a LABCONCO rotary vane 117 vacuum pump) Mass spectrometer (ThermoFisher, Linear Trap Quadrupole Orbitrap Xcalibur 2.0 DS) NMR spectrometer (Bruker) NMR tubes (Fisher Scientific, 16800555) 50 mL conical tubes (ThermoFisher, cat. no. 12-565-271) 500 mL chromatography column 40 mm inner diameter (Fisher Scientific, cat. no. 31-500-913) Temperature-controlled shaking incubator (ThermoFisher, Model MaxQ 4000) NanoDrop spectrophotometer (ThermoFisher Model 2000C) PCR tubes (Fisher Scientific, cat. no. 14230210) Thermocyler (Bioer LifeECO) Sterile syringes, filters, needles (Fisher Scientific) Microcentrifuge tubes (BrandTech, cat. no. 780420) Shaker flasks (PYREX, cat. no. 4446500) Tabletop centrifuge (Thomas Scientific, cat. no. 75007213) Floor centrifuge (Beckman Coulter) pH meter (Fisher Scientific, cat. no. 13636XL150) Sonicator (ThermoFisher Model 505 Soni Dismembrator) CAUTION Sonicator should be in a soundproof box or ear protection should be worn. .. SDS–PAGE system (BioRad) Scanner (Azure Model 600 AZI600) Vials for LC–MS (Waters, cat. no. 186000385C) LC–MS (AB Sciex 5600 QTOF) Glass slides, 75 mm × 25 mm (Chemglass Life Sciences, cat. no. CGQ064001) Silicone rubber spacer (0.5 mm thick) (McMaster-Carr, cat. no 87315K61) 100 mm Petri dish (ThermoFisher, cat. no. FB0875712) Positive displacement pipette (Gilson, cat. no. FD10002) Zeba Spin Desalting Column (ThermoFisher, cat. no PI89882) Radiometer (series 9811–50; Cole-Parmer) UV light source (Lumen Dynamics OmniCure S1500 Spot UV Curing system with an internal 365 nm filter) Chrome photomask (PhotoSciences) Multiphoton microscope (ThorLabs Bergamo II) Standard equipment for mammalian cell culture Biological safety cabinet (Thermo Scientific, cat. no. 1385) CO 2 cell culture incubator set to 37 °C with 5% CO 2 (ThermoFisher) Water bath set to 37 °C (ThermoFisher) Centrifuge (Fisher Scientific, cat. no. 01257134) T25 flasks (Fisher Scientific, cat. no. 12565351) Hemacytometer (Hausser Scientific, cat. no. 3520) 35 mm glass-bottom dishes, 14 mm glass microwell size (Cellvis, cat. no. NC0794151) Autodesk Netfabb Premium 2023.1 ( https://www.autodesk.com ) MATLAB R2019a ( https://www.mathworks.com/products/matlab.html ) Saturated NaCl solution: dissolve 36 g of NaCl in 100 mL H 2 O Brine solution: mix 100 mL of H 2 O with 20 mL of saturated NaCl solution 4 M HCl in 1,4-dioxane: slowly add 10 mL 12 M HCl to 20 mL 1,4-dioxane in a fume hood Carbenicillin (100 mg mL −1 ): dissolve 1 g carbenicillin in 10 mL H 2 O and filter sterilize.
Article Title: Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.
Article Snippet: The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger.. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity.. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide–protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly.
Cell Culture:Article Title: Limited Experimental Susceptibility of Post-Smolt Atlantic salmon (Salmo salar) to an Emergent Strain of Vibrio Anguillarum Serotype O3
Article Snippet: .. Cryopreserved V. anguillarum SO3 isolate Va-21-5-24b (also known as Va210524B) from menhaden collected from the Navesink River, New Jersey, USA, previously shown to have experimental high-dose pathogenicity in Atlantic salmon [9], was streaked onto tryptic soy agar with 5% sheep blood and 1.5% NaCl (BA; Northeast Laboratory Services) and allowed to grow at 22 ± 1 ◦C for 24 h. A single colony was transferred to 250 mL of trypticase soy broth + 1.5% NaCl (TSB, Becton Dickinson and Fisher Scientific) and cultured under aerobic conditions for 24 h at 22 ± 1 ◦C on a magnetic stir plate (Fisher Scientific). .. When the culture reached an optical density of 2.00 (600 nm wavelength) as determined using an Ultraspec 10 Cell Density Meter (Biochrom), a portion was Gram stained and re-cultured on BA to ensure purity.
other:Article Title: Methods of mixing donor human milk during bottling results in fat differences between samples within a pool.
Article Snippet: The influence of milk-banking processes on nutrients in donor human milk (DHM) is largely unknown.. Previous studies have measured nutrients between pools of DHM, but within-pool nutrient differences (between bottles from the same pool) have yet to be elucidated.. The objective of this study was to gain a better understanding of the effect of different mixing characteristics on the distribution of fat, protein, IgA, and lysozyme in bottled, raw DHM.
DNA Synthesis:Article Title: Derivatization of Solid Supports Using a Carbamate‐Linked Tether Containing a Disulfide Functional Group
Article Snippet: .. 2‐[[2‐[bis(4‐methoxyphenyl)phenylmethoxy]ethyl]dithio]ethanol, as per literature (Lack et al., ) or commercial sources (Acinopeptide Co, cat. no. A124685365SN) Acetonitrile (ACN), DNA synthesis grade (Fisher Scientific, cat. no. A998‐4) Pyridine, reagent grade (Sigma‐Aldrich, cat. no. 360570100ML) N,N′ ‐disuccinimidyl carbonate (DSC) (AK Scientific, cat. no. D050) Ethyl acetate (EtOAc) (Sigma‐Aldrich, cat. no. 319902‐4L) Hexane, mixture of isomers (Sigma‐Aldrich, cat. no. 29353‐4L) Sodium bicarbonate saturated solution (see recipe) NaCl saturated brine solution (see recipe) Sodium sulfate, anhydrous (Fisher Chemical, cat. no. S421‐3) Chloroform (Sigma‐Aldrich, cat. no. 366927‐4L) Triethylamine (Fisher Scientific, cat. no. O4885‐1) Dimethylformamide (DMF) (Sigma‐Aldrich, cat. no. 319937) Acetone (Sigma‐Aldrich, cat. no. 179124‐4L) 250‐ml, short neck round–bottom flask, 24/40 (Stony Lab, cat. no. SL000663) White rubber septum, 19/22 (Sigma‐Aldrich, cat. no. Z553972) Magnetic stir bar (Fisher Scientific, cat. no. 14‐513‐62) Magnetic stir plate (Fisher Scientific, cat. no. FB30786160) Glass Pasteur pipette (Fisherbrand, cat. no. 13‐678‐20C) 2.0‐ml Eppendorf microcentrifuge tubes (Fisherbrand, cat. no. 05‐408‐138) Microcentrifuge tubes, 2.0‐ml free standing screw cap tube with cap & O‐ring (Fisherbrand, cat. no. 02‐682‐558) TLC aluminum sheets, Silica gel 60 F 254 (MilliporeSigma, cat. no. 1.05554.0001) 250‐ml separatory funnel (StonyLab, cat. no. SL000874) 250‐ml, Erlenmeyer flask (Fisherbrand, cat. no. FB500250) Mandel rotary evaporator DLAB (Mandel, cat. no. RE100‐S) Rotary vane vacuum pump (Edwards Vacuum, cat. no. E2M2) 4‐Å activated molecular sieves (Sigma‐Aldrich, cat. no. 2086040) Native Amino LCAA CPG 500 Å (ChemGenes, cat. no. N‐5100‐05) Mettler analytical balance AE260 DeltaRange (Mettler Toledo) Weighing paper, 3 × 3–in. (Fisher Scientific, cat. no. 09‐898‐12A) 4‐dram vial (Fisherbrand, cat. no. 03‐338E) 37 ° C heating sand bath 10‐ml disposable syringes (BD, cat. no. 302995) 18‐G disposable needles (BD, cat. no. 305196) TentaGel S‐NH 2 resin SS, 130 μM, 1 % DVB (Advanced Chemtech, cat. no. SJ6011) ..
Transferring:Article Title: Derivatization of Solid Supports Using a Carbamate‐Linked Tether Containing a Disulfide Functional Group
Article Snippet: .. 2‐[[2‐[bis(4‐methoxyphenyl)phenylmethoxy]ethyl]dithio]ethanol, as per literature (Lack et al., ) or commercial sources (Acinopeptide Co, cat. no. A124685365SN) Acetonitrile (ACN), DNA synthesis grade (Fisher Scientific, cat. no. A998‐4) Pyridine, reagent grade (Sigma‐Aldrich, cat. no. 360570100ML) N,N′ ‐disuccinimidyl carbonate (DSC) (AK Scientific, cat. no. D050) Ethyl acetate (EtOAc) (Sigma‐Aldrich, cat. no. 319902‐4L) Hexane, mixture of isomers (Sigma‐Aldrich, cat. no. 29353‐4L) Sodium bicarbonate saturated solution (see recipe) NaCl saturated brine solution (see recipe) Sodium sulfate, anhydrous (Fisher Chemical, cat. no. S421‐3) Chloroform (Sigma‐Aldrich, cat. no. 366927‐4L) Triethylamine (Fisher Scientific, cat. no. O4885‐1) Dimethylformamide (DMF) (Sigma‐Aldrich, cat. no. 319937) Acetone (Sigma‐Aldrich, cat. no. 179124‐4L) 250‐ml, short neck round–bottom flask, 24/40 (Stony Lab, cat. no. SL000663) White rubber septum, 19/22 (Sigma‐Aldrich, cat. no. Z553972) Magnetic stir bar (Fisher Scientific, cat. no. 14‐513‐62) Magnetic stir plate (Fisher Scientific, cat. no. FB30786160) Glass Pasteur pipette (Fisherbrand, cat. no. 13‐678‐20C) 2.0‐ml Eppendorf microcentrifuge tubes (Fisherbrand, cat. no. 05‐408‐138) Microcentrifuge tubes, 2.0‐ml free standing screw cap tube with cap & O‐ring (Fisherbrand, cat. no. 02‐682‐558) TLC aluminum sheets, Silica gel 60 F 254 (MilliporeSigma, cat. no. 1.05554.0001) 250‐ml separatory funnel (StonyLab, cat. no. SL000874) 250‐ml, Erlenmeyer flask (Fisherbrand, cat. no. FB500250) Mandel rotary evaporator DLAB (Mandel, cat. no. RE100‐S) Rotary vane vacuum pump (Edwards Vacuum, cat. no. E2M2) 4‐Å activated molecular sieves (Sigma‐Aldrich, cat. no. 2086040) Native Amino LCAA CPG 500 Å (ChemGenes, cat. no. N‐5100‐05) Mettler analytical balance AE260 DeltaRange (Mettler Toledo) Weighing paper, 3 × 3–in. (Fisher Scientific, cat. no. 09‐898‐12A) 4‐dram vial (Fisherbrand, cat. no. 03‐338E) 37 ° C heating sand bath 10‐ml disposable syringes (BD, cat. no. 302995) 18‐G disposable needles (BD, cat. no. 305196) TentaGel S‐NH 2 resin SS, 130 μM, 1 % DVB (Advanced Chemtech, cat. no. SJ6011) ..
Thin Layer Chromatography:Article Title: Derivatization of Solid Supports Using a Carbamate‐Linked Tether Containing a Disulfide Functional Group
Article Snippet: .. 2‐[[2‐[bis(4‐methoxyphenyl)phenylmethoxy]ethyl]dithio]ethanol, as per literature (Lack et al., ) or commercial sources (Acinopeptide Co, cat. no. A124685365SN) Acetonitrile (ACN), DNA synthesis grade (Fisher Scientific, cat. no. A998‐4) Pyridine, reagent grade (Sigma‐Aldrich, cat. no. 360570100ML) N,N′ ‐disuccinimidyl carbonate (DSC) (AK Scientific, cat. no. D050) Ethyl acetate (EtOAc) (Sigma‐Aldrich, cat. no. 319902‐4L) Hexane, mixture of isomers (Sigma‐Aldrich, cat. no. 29353‐4L) Sodium bicarbonate saturated solution (see recipe) NaCl saturated brine solution (see recipe) Sodium sulfate, anhydrous (Fisher Chemical, cat. no. S421‐3) Chloroform (Sigma‐Aldrich, cat. no. 366927‐4L) Triethylamine (Fisher Scientific, cat. no. O4885‐1) Dimethylformamide (DMF) (Sigma‐Aldrich, cat. no. 319937) Acetone (Sigma‐Aldrich, cat. no. 179124‐4L) 250‐ml, short neck round–bottom flask, 24/40 (Stony Lab, cat. no. SL000663) White rubber septum, 19/22 (Sigma‐Aldrich, cat. no. Z553972) Magnetic stir bar (Fisher Scientific, cat. no. 14‐513‐62) Magnetic stir plate (Fisher Scientific, cat. no. FB30786160) Glass Pasteur pipette (Fisherbrand, cat. no. 13‐678‐20C) 2.0‐ml Eppendorf microcentrifuge tubes (Fisherbrand, cat. no. 05‐408‐138) Microcentrifuge tubes, 2.0‐ml free standing screw cap tube with cap & O‐ring (Fisherbrand, cat. no. 02‐682‐558) TLC aluminum sheets, Silica gel 60 F 254 (MilliporeSigma, cat. no. 1.05554.0001) 250‐ml separatory funnel (StonyLab, cat. no. SL000874) 250‐ml, Erlenmeyer flask (Fisherbrand, cat. no. FB500250) Mandel rotary evaporator DLAB (Mandel, cat. no. RE100‐S) Rotary vane vacuum pump (Edwards Vacuum, cat. no. E2M2) 4‐Å activated molecular sieves (Sigma‐Aldrich, cat. no. 2086040) Native Amino LCAA CPG 500 Å (ChemGenes, cat. no. N‐5100‐05) Mettler analytical balance AE260 DeltaRange (Mettler Toledo) Weighing paper, 3 × 3–in. (Fisher Scientific, cat. no. 09‐898‐12A) 4‐dram vial (Fisherbrand, cat. no. 03‐338E) 37 ° C heating sand bath 10‐ml disposable syringes (BD, cat. no. 302995) 18‐G disposable needles (BD, cat. no. 305196) TentaGel S‐NH 2 resin SS, 130 μM, 1 % DVB (Advanced Chemtech, cat. no. SJ6011) ..
Article Title: A Facile Strategy for 3'-Terminal Functionalization of DNA and RNA via On-Column Conjugation with Integration into Tandem Oligonucleotide Synthesis.
Article Snippet: The following reagents are needed to make a mixed DNA oligonucleotide: 3% (w/v) trichloroacetic acid in dichloromethane (DCM; ChemGenes, cat. no. RN-1462); cap A (acetic anhydride/pyridine/tetrahydrofuran [THF]; ChemGenes, cat. no. RN-1458); cap B (10% N-methylimidazole in THF; ChemGenes, cat. no. RN-1481); activation reagent (0.25 M of 5-ethylthio-tetrazole in ACN; ChemGenes, cat. no. RN1466); oxidation solution (0.02 M iodine/pyridine/H2O/THF; ChemGenes, cat. no. RN-1455); acetonitrile (ACN), DNA synthesis grade (Fisher Scientific, cat. no. A998-4); 2′-deoxyadenosine (n-bz) CED phosphoramidite (ChemGenes, cat. no. ANP-5551); 2′-deoxyguanosine (n-ibu) CED phosphoramidite (ChemGenes, cat. no. ANP-5553); 2′-deoxycytidine (n-acetyl) CED phosphoramidite (ChemGenes, cat. no. ANP-5560); and thymidine CED phosphoramidite (ChemGenes, cat. no. ANP-5554). .. 100 mM sodium acetate buffer (see recipe) 1:1 (v/v) ACN/Milli-Q-purified water Milli-Q-purified water 100-ml short-neck round-bottom flask, 24/40 (StonyLab, cat. no. SL000661) Magnetic stir bar (Fisher Scientific, cat. no. 14-513-62) White rubber septum, 19/22 (Sigma-Aldrich, cat. no. Z553972) Magnetic stir plate (Fisher Scientific, cat. no. FB30786160) Glass Pasteur pipet (Fisher Scientific, cat. no. 13-678-20C) 2.0-ml microcentrifuge tubes (Fisherbrand, cat. no. 05-408-138) TLC aluminum sheets, silica gel 60 F254 (MilliporeSigma, cat. no. 1.05554.0001) Mandel rotary evaporator DLAB (Mandel Scientific Company, cat. no. RE100-S) Rotary vane vacuum pump (Edwards, cat. no. E2M2) 250-ml separatory funnel (StonyLab, cat. no. SL000874) 250-ml Erlenmeyer flask (Fisherbrand, cat. no. FB500250) 250-ml round-bottom flask, 24/40 (StonyLab, cat. no. SL000663) Automatic dispensing pipet UV lamp, 254 nm Mettler analytical balance AE260 DeltaRange (Mettler-Toledo International) Weighing paper, 3 × 3 in. (Fisher Scientific, cat. no. 09-898-12A) Saraya and O’Flaherty 5 of 26 Current Protocols 26911299, 2026, 5, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.70374 by N at Prov Indonesia, W iley O nline L ibrary on [14/05/2026]. .. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Empty synthesis columns, screw type-ABI/Expedite 1.0 μmol (ChemGenes, cat. no. E-1007-ST) 3-ml syringes, Luer-Lok (Cardinal Health Canada, cat. no. BD309657) 18-G disposable needles (Becton Dickinson, cat. no. 305196) Sharps waste container (Cardinal Health Canada, cat. no. 333-040) 10-ml disposable syringes (Becton Dickinson, cat. no. 302995) 1-ml syringes, slip tip (Cardinal Health Canada, cat. no. BD309659) CPR II CPG (Lumiprobe, cat. no. 46860) Automated nucleic acid synthesizer (ABI 394, Applied Biosystems) Vortex mixer (Fisherbrand, cat. no. 02-215-414) 65°C heating sand bath 2.0-ml free-standing screw-cap microcentrifuge tubes with caps and O-rings (Fisherbrand, cat. no. 02-682-558) DNA concentrator, Savant SC 110A SpeedVac Plus (Thermo Fisher Scientific, model no. SC110A-115) High-performance liquid chromatography (HPLC) instrument (Vanquish Core Standard HPLC System, Thermo Fisher Scientific) DNA Pac 200 strong anion exchange (SAX) HPLC column (Thermo Scientific, cat. no. 063000) C18 desalt columns (Sep-Pak, Waters, cat. no. WAT051910) Thermo Fisher Scientific LCQ Fleet Ion Trap with Ion Max-S Source Housing and Ion Max ESI Source (Thermo Fisher Scientific) Synthesis of DMTr thymidine succinimide (1) 1.
Mass Measurement:Article Title: Derivatization of Solid Supports Using a Carbamate‐Linked Tether Containing a Disulfide Functional Group
Article Snippet: .. 2‐[[2‐[bis(4‐methoxyphenyl)phenylmethoxy]ethyl]dithio]ethanol, as per literature (Lack et al., ) or commercial sources (Acinopeptide Co, cat. no. A124685365SN) Acetonitrile (ACN), DNA synthesis grade (Fisher Scientific, cat. no. A998‐4) Pyridine, reagent grade (Sigma‐Aldrich, cat. no. 360570100ML) N,N′ ‐disuccinimidyl carbonate (DSC) (AK Scientific, cat. no. D050) Ethyl acetate (EtOAc) (Sigma‐Aldrich, cat. no. 319902‐4L) Hexane, mixture of isomers (Sigma‐Aldrich, cat. no. 29353‐4L) Sodium bicarbonate saturated solution (see recipe) NaCl saturated brine solution (see recipe) Sodium sulfate, anhydrous (Fisher Chemical, cat. no. S421‐3) Chloroform (Sigma‐Aldrich, cat. no. 366927‐4L) Triethylamine (Fisher Scientific, cat. no. O4885‐1) Dimethylformamide (DMF) (Sigma‐Aldrich, cat. no. 319937) Acetone (Sigma‐Aldrich, cat. no. 179124‐4L) 250‐ml, short neck round–bottom flask, 24/40 (Stony Lab, cat. no. SL000663) White rubber septum, 19/22 (Sigma‐Aldrich, cat. no. Z553972) Magnetic stir bar (Fisher Scientific, cat. no. 14‐513‐62) Magnetic stir plate (Fisher Scientific, cat. no. FB30786160) Glass Pasteur pipette (Fisherbrand, cat. no. 13‐678‐20C) 2.0‐ml Eppendorf microcentrifuge tubes (Fisherbrand, cat. no. 05‐408‐138) Microcentrifuge tubes, 2.0‐ml free standing screw cap tube with cap & O‐ring (Fisherbrand, cat. no. 02‐682‐558) TLC aluminum sheets, Silica gel 60 F 254 (MilliporeSigma, cat. no. 1.05554.0001) 250‐ml separatory funnel (StonyLab, cat. no. SL000874) 250‐ml, Erlenmeyer flask (Fisherbrand, cat. no. FB500250) Mandel rotary evaporator DLAB (Mandel, cat. no. RE100‐S) Rotary vane vacuum pump (Edwards Vacuum, cat. no. E2M2) 4‐Å activated molecular sieves (Sigma‐Aldrich, cat. no. 2086040) Native Amino LCAA CPG 500 Å (ChemGenes, cat. no. N‐5100‐05) Mettler analytical balance AE260 DeltaRange (Mettler Toledo) Weighing paper, 3 × 3–in. (Fisher Scientific, cat. no. 09‐898‐12A) 4‐dram vial (Fisherbrand, cat. no. 03‐338E) 37 ° C heating sand bath 10‐ml disposable syringes (BD, cat. no. 302995) 18‐G disposable needles (BD, cat. no. 305196) TentaGel S‐NH 2 resin SS, 130 μM, 1 % DVB (Advanced Chemtech, cat. no. SJ6011) ..
Article Title: A Facile Strategy for 3'-Terminal Functionalization of DNA and RNA via On-Column Conjugation with Integration into Tandem Oligonucleotide Synthesis.
Article Snippet: The following reagents are needed to make a mixed DNA oligonucleotide: 3% (w/v) trichloroacetic acid in dichloromethane (DCM; ChemGenes, cat. no. RN-1462); cap A (acetic anhydride/pyridine/tetrahydrofuran [THF]; ChemGenes, cat. no. RN-1458); cap B (10% N-methylimidazole in THF; ChemGenes, cat. no. RN-1481); activation reagent (0.25 M of 5-ethylthio-tetrazole in ACN; ChemGenes, cat. no. RN1466); oxidation solution (0.02 M iodine/pyridine/H2O/THF; ChemGenes, cat. no. RN-1455); acetonitrile (ACN), DNA synthesis grade (Fisher Scientific, cat. no. A998-4); 2′-deoxyadenosine (n-bz) CED phosphoramidite (ChemGenes, cat. no. ANP-5551); 2′-deoxyguanosine (n-ibu) CED phosphoramidite (ChemGenes, cat. no. ANP-5553); 2′-deoxycytidine (n-acetyl) CED phosphoramidite (ChemGenes, cat. no. ANP-5560); and thymidine CED phosphoramidite (ChemGenes, cat. no. ANP-5554). .. 100 mM sodium acetate buffer (see recipe) 1:1 (v/v) ACN/Milli-Q-purified water Milli-Q-purified water 100-ml short-neck round-bottom flask, 24/40 (StonyLab, cat. no. SL000661) Magnetic stir bar (Fisher Scientific, cat. no. 14-513-62) White rubber septum, 19/22 (Sigma-Aldrich, cat. no. Z553972) Magnetic stir plate (Fisher Scientific, cat. no. FB30786160) Glass Pasteur pipet (Fisher Scientific, cat. no. 13-678-20C) 2.0-ml microcentrifuge tubes (Fisherbrand, cat. no. 05-408-138) TLC aluminum sheets, silica gel 60 F254 (MilliporeSigma, cat. no. 1.05554.0001) Mandel rotary evaporator DLAB (Mandel Scientific Company, cat. no. RE100-S) Rotary vane vacuum pump (Edwards, cat. no. E2M2) 250-ml separatory funnel (StonyLab, cat. no. SL000874) 250-ml Erlenmeyer flask (Fisherbrand, cat. no. FB500250) 250-ml round-bottom flask, 24/40 (StonyLab, cat. no. SL000663) Automatic dispensing pipet UV lamp, 254 nm Mettler analytical balance AE260 DeltaRange (Mettler-Toledo International) Weighing paper, 3 × 3 in. (Fisher Scientific, cat. no. 09-898-12A) Saraya and O’Flaherty 5 of 26 Current Protocols 26911299, 2026, 5, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.70374 by N at Prov Indonesia, W iley O nline L ibrary on [14/05/2026]. .. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Empty synthesis columns, screw type-ABI/Expedite 1.0 μmol (ChemGenes, cat. no. E-1007-ST) 3-ml syringes, Luer-Lok (Cardinal Health Canada, cat. no. BD309657) 18-G disposable needles (Becton Dickinson, cat. no. 305196) Sharps waste container (Cardinal Health Canada, cat. no. 333-040) 10-ml disposable syringes (Becton Dickinson, cat. no. 302995) 1-ml syringes, slip tip (Cardinal Health Canada, cat. no. BD309659) CPR II CPG (Lumiprobe, cat. no. 46860) Automated nucleic acid synthesizer (ABI 394, Applied Biosystems) Vortex mixer (Fisherbrand, cat. no. 02-215-414) 65°C heating sand bath 2.0-ml free-standing screw-cap microcentrifuge tubes with caps and O-rings (Fisherbrand, cat. no. 02-682-558) DNA concentrator, Savant SC 110A SpeedVac Plus (Thermo Fisher Scientific, model no. SC110A-115) High-performance liquid chromatography (HPLC) instrument (Vanquish Core Standard HPLC System, Thermo Fisher Scientific) DNA Pac 200 strong anion exchange (SAX) HPLC column (Thermo Scientific, cat. no. 063000) C18 desalt columns (Sep-Pak, Waters, cat. no. WAT051910) Thermo Fisher Scientific LCQ Fleet Ion Trap with Ion Max-S Source Housing and Ion Max ESI Source (Thermo Fisher Scientific) Synthesis of DMTr thymidine succinimide (1) 1.
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