Radio Immunoprecipitation:Article Title: Sensing of extracellular l-proline availability by the integrated stress response determines the outcome of cell competition.
Article Snippet: TUNEL staining was performed using In Situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol, as previously described (16). .. Cell lysates were collected using radioimmunoprecipitation assay (RIPA) buffer [50 mM tris- HCl at pH 6.8, 1% sodium deoxycholate 150 mM sodium chloride, 1% NP- 40, 1 mM EDTA, 1× phosphatase inhibitor (Roche), and 1× protease inhibitor (Roche)], quantified using the bicinchoninic acid (BCA) assay quantification (Thermo Fisher Scientific), and resolved using Criterion XT precast gels (BioRad), followed by transfer to polyvinylidene difluoride membranes. .. Antibodies used are as follows: p- eIF2α (1:1000), rabbit polyclonal [Cell Signaling Technology (CST), 9721]; ATF4 (1:1000), rabbit monoclonal (CST, 11,815), CHOP (1:500), mouse monoclonal (sc7351), cleaved caspase 3 (1:1000), rabbit polyclonal (SCT, 9661), proliferating cell nuclear antigen (PCNA; 1:5000), mouse monoclonal (sc- 56), β- actin (1:2500), and rabbit polyclonal (CST, 4967).
Article Title: Sensing of extracellular l -proline availability by the integrated stress response determines the outcome of cell competition
Article Snippet: TUNEL staining was performed using In Situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol, as previously described ( ). .. Cell lysates were collected using radioimmunoprecipitation assay (RIPA) buffer [50 mM tris-HCl at pH 6.8, 1% sodium deoxycholate 150 mM sodium chloride, 1% NP-40, 1 mM EDTA, 1× phosphatase inhibitor (Roche), and 1× protease inhibitor (Roche)], quantified using the bicinchoninic acid (BCA) assay quantification (Thermo Fisher Scientific), and resolved using Criterion XT precast gels (Bio-Rad), followed by transfer to polyvinylidene difluoride membranes. .. Antibodies used are as follows: p-eIF2α (1:1000), rabbit polyclonal [Cell Signaling Technology (CST), 9721]; ATF4 (1:1000), rabbit monoclonal (CST, 11,815), CHOP (1:500), mouse monoclonal (sc-7351), cleaved caspase 3 (1:1000), rabbit polyclonal (SCT, 9661), proliferating cell nuclear antigen (PCNA; 1:5000), mouse monoclonal (sc-56), β-actin (1:2500), and rabbit polyclonal (CST, 4967).
Protease Inhibitor:Article Title: Sensing of extracellular l-proline availability by the integrated stress response determines the outcome of cell competition.
Article Snippet: TUNEL staining was performed using In Situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol, as previously described (16). .. Cell lysates were collected using radioimmunoprecipitation assay (RIPA) buffer [50 mM tris- HCl at pH 6.8, 1% sodium deoxycholate 150 mM sodium chloride, 1% NP- 40, 1 mM EDTA, 1× phosphatase inhibitor (Roche), and 1× protease inhibitor (Roche)], quantified using the bicinchoninic acid (BCA) assay quantification (Thermo Fisher Scientific), and resolved using Criterion XT precast gels (BioRad), followed by transfer to polyvinylidene difluoride membranes. .. Antibodies used are as follows: p- eIF2α (1:1000), rabbit polyclonal [Cell Signaling Technology (CST), 9721]; ATF4 (1:1000), rabbit monoclonal (CST, 11,815), CHOP (1:500), mouse monoclonal (sc7351), cleaved caspase 3 (1:1000), rabbit polyclonal (SCT, 9661), proliferating cell nuclear antigen (PCNA; 1:5000), mouse monoclonal (sc- 56), β- actin (1:2500), and rabbit polyclonal (CST, 4967).
Article Title: Sensing of extracellular l -proline availability by the integrated stress response determines the outcome of cell competition
Article Snippet: TUNEL staining was performed using In Situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol, as previously described ( ). .. Cell lysates were collected using radioimmunoprecipitation assay (RIPA) buffer [50 mM tris-HCl at pH 6.8, 1% sodium deoxycholate 150 mM sodium chloride, 1% NP-40, 1 mM EDTA, 1× phosphatase inhibitor (Roche), and 1× protease inhibitor (Roche)], quantified using the bicinchoninic acid (BCA) assay quantification (Thermo Fisher Scientific), and resolved using Criterion XT precast gels (Bio-Rad), followed by transfer to polyvinylidene difluoride membranes. .. Antibodies used are as follows: p-eIF2α (1:1000), rabbit polyclonal [Cell Signaling Technology (CST), 9721]; ATF4 (1:1000), rabbit monoclonal (CST, 11,815), CHOP (1:500), mouse monoclonal (sc-7351), cleaved caspase 3 (1:1000), rabbit polyclonal (SCT, 9661), proliferating cell nuclear antigen (PCNA; 1:5000), mouse monoclonal (sc-56), β-actin (1:2500), and rabbit polyclonal (CST, 4967).
BIA-KA:Article Title: Sensing of extracellular l-proline availability by the integrated stress response determines the outcome of cell competition.
Article Snippet: TUNEL staining was performed using In Situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol, as previously described (16). .. Cell lysates were collected using radioimmunoprecipitation assay (RIPA) buffer [50 mM tris- HCl at pH 6.8, 1% sodium deoxycholate 150 mM sodium chloride, 1% NP- 40, 1 mM EDTA, 1× phosphatase inhibitor (Roche), and 1× protease inhibitor (Roche)], quantified using the bicinchoninic acid (BCA) assay quantification (Thermo Fisher Scientific), and resolved using Criterion XT precast gels (BioRad), followed by transfer to polyvinylidene difluoride membranes. .. Antibodies used are as follows: p- eIF2α (1:1000), rabbit polyclonal [Cell Signaling Technology (CST), 9721]; ATF4 (1:1000), rabbit monoclonal (CST, 11,815), CHOP (1:500), mouse monoclonal (sc7351), cleaved caspase 3 (1:1000), rabbit polyclonal (SCT, 9661), proliferating cell nuclear antigen (PCNA; 1:5000), mouse monoclonal (sc- 56), β- actin (1:2500), and rabbit polyclonal (CST, 4967).
Article Title: Sensing of extracellular l -proline availability by the integrated stress response determines the outcome of cell competition
Article Snippet: TUNEL staining was performed using In Situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol, as previously described ( ). .. Cell lysates were collected using radioimmunoprecipitation assay (RIPA) buffer [50 mM tris-HCl at pH 6.8, 1% sodium deoxycholate 150 mM sodium chloride, 1% NP-40, 1 mM EDTA, 1× phosphatase inhibitor (Roche), and 1× protease inhibitor (Roche)], quantified using the bicinchoninic acid (BCA) assay quantification (Thermo Fisher Scientific), and resolved using Criterion XT precast gels (Bio-Rad), followed by transfer to polyvinylidene difluoride membranes. .. Antibodies used are as follows: p-eIF2α (1:1000), rabbit polyclonal [Cell Signaling Technology (CST), 9721]; ATF4 (1:1000), rabbit monoclonal (CST, 11,815), CHOP (1:500), mouse monoclonal (sc-7351), cleaved caspase 3 (1:1000), rabbit polyclonal (SCT, 9661), proliferating cell nuclear antigen (PCNA; 1:5000), mouse monoclonal (sc-56), β-actin (1:2500), and rabbit polyclonal (CST, 4967).
SDS Page:Article Title: Liver-targeted Angptl4 silencing by antisense oligonucleotide treatment attenuates hyperlipidaemia and atherosclerosis development in APOE*3-Leiden.CETP mice
Article Snippet: .. For this, proteins were separated by SDS-PAGE using Criterion XT Precast gels (Bio-Rad, Hercules, CA, USA) and transferred to the polyvinylidene difluoride membrane (Immobilon-P; Millipore, Billerica, MA, USA). .. For this, proteins were separated by SDS-PAGE using Criterion XT Precast gels (Bio-Rad, Hercules, CA, USA) and transferred to the polyvinylidene difluoride membrane (Immobilon-P; Millipore, Billerica, MA, USA).
Article Title: Endothelial-pericyte interactions activate insulin signaling and its implications for blood-brain barrier dysfunction in Alzheimer’s disease
Article Snippet: Total protein concentrations in the lysates were determined by bicinchoninic acid (BCA) assay (Pierce, Waltham, MA). .. The lysates (25 μg protein per lane) were loaded onto 4-12% Criterion XT precast gels and proteins were separated by SDS-PAGE under reducing conditions (Bio-Rad Laboratories, Hercules, CA). .. The proteins were then electroblotted onto a 0.45 μm nitrocellulose membrane, blocked with 5% nonfat dry milk protein (Bio-Rad Laboratories, Hercules, CA), and incubated overnight at 4 °C with primary antibodies against TIMP1 (1:500, Cell Signaling Technology, Danvers, MA), Vinculin (1:2000, Cell Signaling Technology, Danvers, MA), p-AKT (1:1000, Cell Signaling Technology, Danvers, MA), p-ERK (1:1000, Cell Signaling Technology, Danvers, MA), total-AKT (1:1000, Cell Signaling Technology, Danvers, MA), total-ERK (1:1000, Cell Signaling Technology, Danvers, MA), GAPDH, claudin-5 (1:1000, Cell Signaling Technology, Danvers, MA) and collagen-IV (1:1000, Thermo Fisher Scientific).
Membrane:Article Title: Liver-targeted Angptl4 silencing by antisense oligonucleotide treatment attenuates hyperlipidaemia and atherosclerosis development in APOE*3-Leiden.CETP mice
Article Snippet: .. For this, proteins were separated by SDS-PAGE using Criterion XT Precast gels (Bio-Rad, Hercules, CA, USA) and transferred to the polyvinylidene difluoride membrane (Immobilon-P; Millipore, Billerica, MA, USA). .. For this, proteins were separated by SDS-PAGE using Criterion XT Precast gels (Bio-Rad, Hercules, CA, USA) and transferred to the polyvinylidene difluoride membrane (Immobilon-P; Millipore, Billerica, MA, USA).
Plasmid Preparation:Article Title: Engineering, Expression, Purification, and Application of Glycosaminoglycan‐Specific Antibodies
Article Snippet: .. E. coli BL21‐CodonPlus (DE3)‐RIL competent cells (Agilent, cat. no. 230245) Plasmid DNA 2×YT medium (see recipe): without antibiotics containing 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose containing 50 μg/ml kanamycin only 1 M IPTG (see recipe) scFv‐H resuspension buffer (see recipe) 50% (v/v) His60 slurry (see recipe), freshly prepared His60 wash buffer (see recipe) His60 elution buffer (see recipe) 4× Laemmli buffer (Bio‐Rad, cat. no. 1610747) Dithiothreitol (DTT, Gold Bio, cat. no. DTT100) Criterion XT precast gels, 4‐12% Bis‐Tris (Bio‐Rad, cat. no. 3450125) Unstained protein standard (New England Biolabs, cat. no. P7717S) SDS‐PAGE running buffer (see recipe) SDS‐PAGE staining solution (see recipe) LB agar plates (see recipe), 8 wells, with 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose 10% (v/v) antifoam solution (see recipe) scFv‐H SEC running buffer (see recipe) Gel filtration standards (Bio‐Rad, cat. no. 1511901) 96‐well PCR plate (Thermo Fisher Scientific, cat. no. AB2396) 42°C heat block or thermocycler for 96‐well plate 96‐well 2‐ml deep‐well blocks (USA Scientific, cat. no. 1896‐2000), sterilized by autoclaving at 121°C for 15 min AeraSeal breathable seals (Excel Scientific, cat. no. B100) Bacterial shaking incubator at 37° and 22°C (e.g., Shel Lab S16R‐HS‐2 for small scale, New Brunswick Scientific I 26 for large scale) Spectrophotometer (e.g., Stunner, Unchained Labs) Bacterial plate incubator (e.g., Shel Lab S16R‐HS‐2) at 37°C Plate shaker (e.g., Ika MTS 2/4) at 4°C 96‐well, 0.22‐μm filter plate (Thomson, cat. no. 931919) Vacuum manifold for 96‐well filter plates (e.g., Sigma, cat. no. 57192‐U) SDS‐PAGE gel running equipment (Bio‐Rad, cat. no. 1656019) Gel imaging system (e.g., FluorChem, ProteinSimple) 250‐ml Erlenmeyer flasks (VWR, cat. no. 76531‐762) 2‐L baffled flasks (Genesis Pyrex, cat. no. 4446‐2L) 1‐L centrifuge bottles (Beckman, cat. no. C31597 ) 50‐ml centrifuge tubes (Fisher Scientific, cat. no. 14955239) Rocker 20‐ml Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 0.45‐μm syringe filters (Genesee Scientific, cat. no. 25‐242) FPLC system for protein purification (e.g., GE ÄKTA) equipped with HiLoad 16/600 Superdex 75‐pg column (Cytiva, cat. no. 28989333) 10‐kDa centrifugal concentrators (Amicon Ultra‐15, Millipore, cat. no. UFC901024) ..
Staining:Article Title: Engineering, Expression, Purification, and Application of Glycosaminoglycan‐Specific Antibodies
Article Snippet: .. E. coli BL21‐CodonPlus (DE3)‐RIL competent cells (Agilent, cat. no. 230245) Plasmid DNA 2×YT medium (see recipe): without antibiotics containing 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose containing 50 μg/ml kanamycin only 1 M IPTG (see recipe) scFv‐H resuspension buffer (see recipe) 50% (v/v) His60 slurry (see recipe), freshly prepared His60 wash buffer (see recipe) His60 elution buffer (see recipe) 4× Laemmli buffer (Bio‐Rad, cat. no. 1610747) Dithiothreitol (DTT, Gold Bio, cat. no. DTT100) Criterion XT precast gels, 4‐12% Bis‐Tris (Bio‐Rad, cat. no. 3450125) Unstained protein standard (New England Biolabs, cat. no. P7717S) SDS‐PAGE running buffer (see recipe) SDS‐PAGE staining solution (see recipe) LB agar plates (see recipe), 8 wells, with 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose 10% (v/v) antifoam solution (see recipe) scFv‐H SEC running buffer (see recipe) Gel filtration standards (Bio‐Rad, cat. no. 1511901) 96‐well PCR plate (Thermo Fisher Scientific, cat. no. AB2396) 42°C heat block or thermocycler for 96‐well plate 96‐well 2‐ml deep‐well blocks (USA Scientific, cat. no. 1896‐2000), sterilized by autoclaving at 121°C for 15 min AeraSeal breathable seals (Excel Scientific, cat. no. B100) Bacterial shaking incubator at 37° and 22°C (e.g., Shel Lab S16R‐HS‐2 for small scale, New Brunswick Scientific I 26 for large scale) Spectrophotometer (e.g., Stunner, Unchained Labs) Bacterial plate incubator (e.g., Shel Lab S16R‐HS‐2) at 37°C Plate shaker (e.g., Ika MTS 2/4) at 4°C 96‐well, 0.22‐μm filter plate (Thomson, cat. no. 931919) Vacuum manifold for 96‐well filter plates (e.g., Sigma, cat. no. 57192‐U) SDS‐PAGE gel running equipment (Bio‐Rad, cat. no. 1656019) Gel imaging system (e.g., FluorChem, ProteinSimple) 250‐ml Erlenmeyer flasks (VWR, cat. no. 76531‐762) 2‐L baffled flasks (Genesis Pyrex, cat. no. 4446‐2L) 1‐L centrifuge bottles (Beckman, cat. no. C31597 ) 50‐ml centrifuge tubes (Fisher Scientific, cat. no. 14955239) Rocker 20‐ml Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 0.45‐μm syringe filters (Genesee Scientific, cat. no. 25‐242) FPLC system for protein purification (e.g., GE ÄKTA) equipped with HiLoad 16/600 Superdex 75‐pg column (Cytiva, cat. no. 28989333) 10‐kDa centrifugal concentrators (Amicon Ultra‐15, Millipore, cat. no. UFC901024) ..
Filtration:Article Title: Engineering, Expression, Purification, and Application of Glycosaminoglycan‐Specific Antibodies
Article Snippet: .. E. coli BL21‐CodonPlus (DE3)‐RIL competent cells (Agilent, cat. no. 230245) Plasmid DNA 2×YT medium (see recipe): without antibiotics containing 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose containing 50 μg/ml kanamycin only 1 M IPTG (see recipe) scFv‐H resuspension buffer (see recipe) 50% (v/v) His60 slurry (see recipe), freshly prepared His60 wash buffer (see recipe) His60 elution buffer (see recipe) 4× Laemmli buffer (Bio‐Rad, cat. no. 1610747) Dithiothreitol (DTT, Gold Bio, cat. no. DTT100) Criterion XT precast gels, 4‐12% Bis‐Tris (Bio‐Rad, cat. no. 3450125) Unstained protein standard (New England Biolabs, cat. no. P7717S) SDS‐PAGE running buffer (see recipe) SDS‐PAGE staining solution (see recipe) LB agar plates (see recipe), 8 wells, with 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose 10% (v/v) antifoam solution (see recipe) scFv‐H SEC running buffer (see recipe) Gel filtration standards (Bio‐Rad, cat. no. 1511901) 96‐well PCR plate (Thermo Fisher Scientific, cat. no. AB2396) 42°C heat block or thermocycler for 96‐well plate 96‐well 2‐ml deep‐well blocks (USA Scientific, cat. no. 1896‐2000), sterilized by autoclaving at 121°C for 15 min AeraSeal breathable seals (Excel Scientific, cat. no. B100) Bacterial shaking incubator at 37° and 22°C (e.g., Shel Lab S16R‐HS‐2 for small scale, New Brunswick Scientific I 26 for large scale) Spectrophotometer (e.g., Stunner, Unchained Labs) Bacterial plate incubator (e.g., Shel Lab S16R‐HS‐2) at 37°C Plate shaker (e.g., Ika MTS 2/4) at 4°C 96‐well, 0.22‐μm filter plate (Thomson, cat. no. 931919) Vacuum manifold for 96‐well filter plates (e.g., Sigma, cat. no. 57192‐U) SDS‐PAGE gel running equipment (Bio‐Rad, cat. no. 1656019) Gel imaging system (e.g., FluorChem, ProteinSimple) 250‐ml Erlenmeyer flasks (VWR, cat. no. 76531‐762) 2‐L baffled flasks (Genesis Pyrex, cat. no. 4446‐2L) 1‐L centrifuge bottles (Beckman, cat. no. C31597 ) 50‐ml centrifuge tubes (Fisher Scientific, cat. no. 14955239) Rocker 20‐ml Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 0.45‐μm syringe filters (Genesee Scientific, cat. no. 25‐242) FPLC system for protein purification (e.g., GE ÄKTA) equipped with HiLoad 16/600 Superdex 75‐pg column (Cytiva, cat. no. 28989333) 10‐kDa centrifugal concentrators (Amicon Ultra‐15, Millipore, cat. no. UFC901024) ..
Polymerase Chain Reaction:Article Title: Engineering, Expression, Purification, and Application of Glycosaminoglycan‐Specific Antibodies
Article Snippet: .. E. coli BL21‐CodonPlus (DE3)‐RIL competent cells (Agilent, cat. no. 230245) Plasmid DNA 2×YT medium (see recipe): without antibiotics containing 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose containing 50 μg/ml kanamycin only 1 M IPTG (see recipe) scFv‐H resuspension buffer (see recipe) 50% (v/v) His60 slurry (see recipe), freshly prepared His60 wash buffer (see recipe) His60 elution buffer (see recipe) 4× Laemmli buffer (Bio‐Rad, cat. no. 1610747) Dithiothreitol (DTT, Gold Bio, cat. no. DTT100) Criterion XT precast gels, 4‐12% Bis‐Tris (Bio‐Rad, cat. no. 3450125) Unstained protein standard (New England Biolabs, cat. no. P7717S) SDS‐PAGE running buffer (see recipe) SDS‐PAGE staining solution (see recipe) LB agar plates (see recipe), 8 wells, with 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose 10% (v/v) antifoam solution (see recipe) scFv‐H SEC running buffer (see recipe) Gel filtration standards (Bio‐Rad, cat. no. 1511901) 96‐well PCR plate (Thermo Fisher Scientific, cat. no. AB2396) 42°C heat block or thermocycler for 96‐well plate 96‐well 2‐ml deep‐well blocks (USA Scientific, cat. no. 1896‐2000), sterilized by autoclaving at 121°C for 15 min AeraSeal breathable seals (Excel Scientific, cat. no. B100) Bacterial shaking incubator at 37° and 22°C (e.g., Shel Lab S16R‐HS‐2 for small scale, New Brunswick Scientific I 26 for large scale) Spectrophotometer (e.g., Stunner, Unchained Labs) Bacterial plate incubator (e.g., Shel Lab S16R‐HS‐2) at 37°C Plate shaker (e.g., Ika MTS 2/4) at 4°C 96‐well, 0.22‐μm filter plate (Thomson, cat. no. 931919) Vacuum manifold for 96‐well filter plates (e.g., Sigma, cat. no. 57192‐U) SDS‐PAGE gel running equipment (Bio‐Rad, cat. no. 1656019) Gel imaging system (e.g., FluorChem, ProteinSimple) 250‐ml Erlenmeyer flasks (VWR, cat. no. 76531‐762) 2‐L baffled flasks (Genesis Pyrex, cat. no. 4446‐2L) 1‐L centrifuge bottles (Beckman, cat. no. C31597 ) 50‐ml centrifuge tubes (Fisher Scientific, cat. no. 14955239) Rocker 20‐ml Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 0.45‐μm syringe filters (Genesee Scientific, cat. no. 25‐242) FPLC system for protein purification (e.g., GE ÄKTA) equipped with HiLoad 16/600 Superdex 75‐pg column (Cytiva, cat. no. 28989333) 10‐kDa centrifugal concentrators (Amicon Ultra‐15, Millipore, cat. no. UFC901024) ..
Blocking Assay:Article Title: Engineering, Expression, Purification, and Application of Glycosaminoglycan‐Specific Antibodies
Article Snippet: .. E. coli BL21‐CodonPlus (DE3)‐RIL competent cells (Agilent, cat. no. 230245) Plasmid DNA 2×YT medium (see recipe): without antibiotics containing 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose containing 50 μg/ml kanamycin only 1 M IPTG (see recipe) scFv‐H resuspension buffer (see recipe) 50% (v/v) His60 slurry (see recipe), freshly prepared His60 wash buffer (see recipe) His60 elution buffer (see recipe) 4× Laemmli buffer (Bio‐Rad, cat. no. 1610747) Dithiothreitol (DTT, Gold Bio, cat. no. DTT100) Criterion XT precast gels, 4‐12% Bis‐Tris (Bio‐Rad, cat. no. 3450125) Unstained protein standard (New England Biolabs, cat. no. P7717S) SDS‐PAGE running buffer (see recipe) SDS‐PAGE staining solution (see recipe) LB agar plates (see recipe), 8 wells, with 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose 10% (v/v) antifoam solution (see recipe) scFv‐H SEC running buffer (see recipe) Gel filtration standards (Bio‐Rad, cat. no. 1511901) 96‐well PCR plate (Thermo Fisher Scientific, cat. no. AB2396) 42°C heat block or thermocycler for 96‐well plate 96‐well 2‐ml deep‐well blocks (USA Scientific, cat. no. 1896‐2000), sterilized by autoclaving at 121°C for 15 min AeraSeal breathable seals (Excel Scientific, cat. no. B100) Bacterial shaking incubator at 37° and 22°C (e.g., Shel Lab S16R‐HS‐2 for small scale, New Brunswick Scientific I 26 for large scale) Spectrophotometer (e.g., Stunner, Unchained Labs) Bacterial plate incubator (e.g., Shel Lab S16R‐HS‐2) at 37°C Plate shaker (e.g., Ika MTS 2/4) at 4°C 96‐well, 0.22‐μm filter plate (Thomson, cat. no. 931919) Vacuum manifold for 96‐well filter plates (e.g., Sigma, cat. no. 57192‐U) SDS‐PAGE gel running equipment (Bio‐Rad, cat. no. 1656019) Gel imaging system (e.g., FluorChem, ProteinSimple) 250‐ml Erlenmeyer flasks (VWR, cat. no. 76531‐762) 2‐L baffled flasks (Genesis Pyrex, cat. no. 4446‐2L) 1‐L centrifuge bottles (Beckman, cat. no. C31597 ) 50‐ml centrifuge tubes (Fisher Scientific, cat. no. 14955239) Rocker 20‐ml Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 0.45‐μm syringe filters (Genesee Scientific, cat. no. 25‐242) FPLC system for protein purification (e.g., GE ÄKTA) equipped with HiLoad 16/600 Superdex 75‐pg column (Cytiva, cat. no. 28989333) 10‐kDa centrifugal concentrators (Amicon Ultra‐15, Millipore, cat. no. UFC901024) ..
Spectrophotometry:Article Title: Engineering, Expression, Purification, and Application of Glycosaminoglycan‐Specific Antibodies
Article Snippet: .. E. coli BL21‐CodonPlus (DE3)‐RIL competent cells (Agilent, cat. no. 230245) Plasmid DNA 2×YT medium (see recipe): without antibiotics containing 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose containing 50 μg/ml kanamycin only 1 M IPTG (see recipe) scFv‐H resuspension buffer (see recipe) 50% (v/v) His60 slurry (see recipe), freshly prepared His60 wash buffer (see recipe) His60 elution buffer (see recipe) 4× Laemmli buffer (Bio‐Rad, cat. no. 1610747) Dithiothreitol (DTT, Gold Bio, cat. no. DTT100) Criterion XT precast gels, 4‐12% Bis‐Tris (Bio‐Rad, cat. no. 3450125) Unstained protein standard (New England Biolabs, cat. no. P7717S) SDS‐PAGE running buffer (see recipe) SDS‐PAGE staining solution (see recipe) LB agar plates (see recipe), 8 wells, with 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose 10% (v/v) antifoam solution (see recipe) scFv‐H SEC running buffer (see recipe) Gel filtration standards (Bio‐Rad, cat. no. 1511901) 96‐well PCR plate (Thermo Fisher Scientific, cat. no. AB2396) 42°C heat block or thermocycler for 96‐well plate 96‐well 2‐ml deep‐well blocks (USA Scientific, cat. no. 1896‐2000), sterilized by autoclaving at 121°C for 15 min AeraSeal breathable seals (Excel Scientific, cat. no. B100) Bacterial shaking incubator at 37° and 22°C (e.g., Shel Lab S16R‐HS‐2 for small scale, New Brunswick Scientific I 26 for large scale) Spectrophotometer (e.g., Stunner, Unchained Labs) Bacterial plate incubator (e.g., Shel Lab S16R‐HS‐2) at 37°C Plate shaker (e.g., Ika MTS 2/4) at 4°C 96‐well, 0.22‐μm filter plate (Thomson, cat. no. 931919) Vacuum manifold for 96‐well filter plates (e.g., Sigma, cat. no. 57192‐U) SDS‐PAGE gel running equipment (Bio‐Rad, cat. no. 1656019) Gel imaging system (e.g., FluorChem, ProteinSimple) 250‐ml Erlenmeyer flasks (VWR, cat. no. 76531‐762) 2‐L baffled flasks (Genesis Pyrex, cat. no. 4446‐2L) 1‐L centrifuge bottles (Beckman, cat. no. C31597 ) 50‐ml centrifuge tubes (Fisher Scientific, cat. no. 14955239) Rocker 20‐ml Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 0.45‐μm syringe filters (Genesee Scientific, cat. no. 25‐242) FPLC system for protein purification (e.g., GE ÄKTA) equipped with HiLoad 16/600 Superdex 75‐pg column (Cytiva, cat. no. 28989333) 10‐kDa centrifugal concentrators (Amicon Ultra‐15, Millipore, cat. no. UFC901024) ..
Imaging:Article Title: Engineering, Expression, Purification, and Application of Glycosaminoglycan‐Specific Antibodies
Article Snippet: .. E. coli BL21‐CodonPlus (DE3)‐RIL competent cells (Agilent, cat. no. 230245) Plasmid DNA 2×YT medium (see recipe): without antibiotics containing 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose containing 50 μg/ml kanamycin only 1 M IPTG (see recipe) scFv‐H resuspension buffer (see recipe) 50% (v/v) His60 slurry (see recipe), freshly prepared His60 wash buffer (see recipe) His60 elution buffer (see recipe) 4× Laemmli buffer (Bio‐Rad, cat. no. 1610747) Dithiothreitol (DTT, Gold Bio, cat. no. DTT100) Criterion XT precast gels, 4‐12% Bis‐Tris (Bio‐Rad, cat. no. 3450125) Unstained protein standard (New England Biolabs, cat. no. P7717S) SDS‐PAGE running buffer (see recipe) SDS‐PAGE staining solution (see recipe) LB agar plates (see recipe), 8 wells, with 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose 10% (v/v) antifoam solution (see recipe) scFv‐H SEC running buffer (see recipe) Gel filtration standards (Bio‐Rad, cat. no. 1511901) 96‐well PCR plate (Thermo Fisher Scientific, cat. no. AB2396) 42°C heat block or thermocycler for 96‐well plate 96‐well 2‐ml deep‐well blocks (USA Scientific, cat. no. 1896‐2000), sterilized by autoclaving at 121°C for 15 min AeraSeal breathable seals (Excel Scientific, cat. no. B100) Bacterial shaking incubator at 37° and 22°C (e.g., Shel Lab S16R‐HS‐2 for small scale, New Brunswick Scientific I 26 for large scale) Spectrophotometer (e.g., Stunner, Unchained Labs) Bacterial plate incubator (e.g., Shel Lab S16R‐HS‐2) at 37°C Plate shaker (e.g., Ika MTS 2/4) at 4°C 96‐well, 0.22‐μm filter plate (Thomson, cat. no. 931919) Vacuum manifold for 96‐well filter plates (e.g., Sigma, cat. no. 57192‐U) SDS‐PAGE gel running equipment (Bio‐Rad, cat. no. 1656019) Gel imaging system (e.g., FluorChem, ProteinSimple) 250‐ml Erlenmeyer flasks (VWR, cat. no. 76531‐762) 2‐L baffled flasks (Genesis Pyrex, cat. no. 4446‐2L) 1‐L centrifuge bottles (Beckman, cat. no. C31597 ) 50‐ml centrifuge tubes (Fisher Scientific, cat. no. 14955239) Rocker 20‐ml Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 0.45‐μm syringe filters (Genesee Scientific, cat. no. 25‐242) FPLC system for protein purification (e.g., GE ÄKTA) equipped with HiLoad 16/600 Superdex 75‐pg column (Cytiva, cat. no. 28989333) 10‐kDa centrifugal concentrators (Amicon Ultra‐15, Millipore, cat. no. UFC901024) ..
Chromatography:Article Title: Engineering, Expression, Purification, and Application of Glycosaminoglycan‐Specific Antibodies
Article Snippet: .. E. coli BL21‐CodonPlus (DE3)‐RIL competent cells (Agilent, cat. no. 230245) Plasmid DNA 2×YT medium (see recipe): without antibiotics containing 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose containing 50 μg/ml kanamycin only 1 M IPTG (see recipe) scFv‐H resuspension buffer (see recipe) 50% (v/v) His60 slurry (see recipe), freshly prepared His60 wash buffer (see recipe) His60 elution buffer (see recipe) 4× Laemmli buffer (Bio‐Rad, cat. no. 1610747) Dithiothreitol (DTT, Gold Bio, cat. no. DTT100) Criterion XT precast gels, 4‐12% Bis‐Tris (Bio‐Rad, cat. no. 3450125) Unstained protein standard (New England Biolabs, cat. no. P7717S) SDS‐PAGE running buffer (see recipe) SDS‐PAGE staining solution (see recipe) LB agar plates (see recipe), 8 wells, with 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose 10% (v/v) antifoam solution (see recipe) scFv‐H SEC running buffer (see recipe) Gel filtration standards (Bio‐Rad, cat. no. 1511901) 96‐well PCR plate (Thermo Fisher Scientific, cat. no. AB2396) 42°C heat block or thermocycler for 96‐well plate 96‐well 2‐ml deep‐well blocks (USA Scientific, cat. no. 1896‐2000), sterilized by autoclaving at 121°C for 15 min AeraSeal breathable seals (Excel Scientific, cat. no. B100) Bacterial shaking incubator at 37° and 22°C (e.g., Shel Lab S16R‐HS‐2 for small scale, New Brunswick Scientific I 26 for large scale) Spectrophotometer (e.g., Stunner, Unchained Labs) Bacterial plate incubator (e.g., Shel Lab S16R‐HS‐2) at 37°C Plate shaker (e.g., Ika MTS 2/4) at 4°C 96‐well, 0.22‐μm filter plate (Thomson, cat. no. 931919) Vacuum manifold for 96‐well filter plates (e.g., Sigma, cat. no. 57192‐U) SDS‐PAGE gel running equipment (Bio‐Rad, cat. no. 1656019) Gel imaging system (e.g., FluorChem, ProteinSimple) 250‐ml Erlenmeyer flasks (VWR, cat. no. 76531‐762) 2‐L baffled flasks (Genesis Pyrex, cat. no. 4446‐2L) 1‐L centrifuge bottles (Beckman, cat. no. C31597 ) 50‐ml centrifuge tubes (Fisher Scientific, cat. no. 14955239) Rocker 20‐ml Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 0.45‐μm syringe filters (Genesee Scientific, cat. no. 25‐242) FPLC system for protein purification (e.g., GE ÄKTA) equipped with HiLoad 16/600 Superdex 75‐pg column (Cytiva, cat. no. 28989333) 10‐kDa centrifugal concentrators (Amicon Ultra‐15, Millipore, cat. no. UFC901024) ..
Fast Protein Liquid Chromatography:Article Title: Engineering, Expression, Purification, and Application of Glycosaminoglycan‐Specific Antibodies
Article Snippet: .. E. coli BL21‐CodonPlus (DE3)‐RIL competent cells (Agilent, cat. no. 230245) Plasmid DNA 2×YT medium (see recipe): without antibiotics containing 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose containing 50 μg/ml kanamycin only 1 M IPTG (see recipe) scFv‐H resuspension buffer (see recipe) 50% (v/v) His60 slurry (see recipe), freshly prepared His60 wash buffer (see recipe) His60 elution buffer (see recipe) 4× Laemmli buffer (Bio‐Rad, cat. no. 1610747) Dithiothreitol (DTT, Gold Bio, cat. no. DTT100) Criterion XT precast gels, 4‐12% Bis‐Tris (Bio‐Rad, cat. no. 3450125) Unstained protein standard (New England Biolabs, cat. no. P7717S) SDS‐PAGE running buffer (see recipe) SDS‐PAGE staining solution (see recipe) LB agar plates (see recipe), 8 wells, with 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose 10% (v/v) antifoam solution (see recipe) scFv‐H SEC running buffer (see recipe) Gel filtration standards (Bio‐Rad, cat. no. 1511901) 96‐well PCR plate (Thermo Fisher Scientific, cat. no. AB2396) 42°C heat block or thermocycler for 96‐well plate 96‐well 2‐ml deep‐well blocks (USA Scientific, cat. no. 1896‐2000), sterilized by autoclaving at 121°C for 15 min AeraSeal breathable seals (Excel Scientific, cat. no. B100) Bacterial shaking incubator at 37° and 22°C (e.g., Shel Lab S16R‐HS‐2 for small scale, New Brunswick Scientific I 26 for large scale) Spectrophotometer (e.g., Stunner, Unchained Labs) Bacterial plate incubator (e.g., Shel Lab S16R‐HS‐2) at 37°C Plate shaker (e.g., Ika MTS 2/4) at 4°C 96‐well, 0.22‐μm filter plate (Thomson, cat. no. 931919) Vacuum manifold for 96‐well filter plates (e.g., Sigma, cat. no. 57192‐U) SDS‐PAGE gel running equipment (Bio‐Rad, cat. no. 1656019) Gel imaging system (e.g., FluorChem, ProteinSimple) 250‐ml Erlenmeyer flasks (VWR, cat. no. 76531‐762) 2‐L baffled flasks (Genesis Pyrex, cat. no. 4446‐2L) 1‐L centrifuge bottles (Beckman, cat. no. C31597 ) 50‐ml centrifuge tubes (Fisher Scientific, cat. no. 14955239) Rocker 20‐ml Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 0.45‐μm syringe filters (Genesee Scientific, cat. no. 25‐242) FPLC system for protein purification (e.g., GE ÄKTA) equipped with HiLoad 16/600 Superdex 75‐pg column (Cytiva, cat. no. 28989333) 10‐kDa centrifugal concentrators (Amicon Ultra‐15, Millipore, cat. no. UFC901024) ..
Protein Purification:Article Title: Engineering, Expression, Purification, and Application of Glycosaminoglycan‐Specific Antibodies
Article Snippet: .. E. coli BL21‐CodonPlus (DE3)‐RIL competent cells (Agilent, cat. no. 230245) Plasmid DNA 2×YT medium (see recipe): without antibiotics containing 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose containing 50 μg/ml kanamycin only 1 M IPTG (see recipe) scFv‐H resuspension buffer (see recipe) 50% (v/v) His60 slurry (see recipe), freshly prepared His60 wash buffer (see recipe) His60 elution buffer (see recipe) 4× Laemmli buffer (Bio‐Rad, cat. no. 1610747) Dithiothreitol (DTT, Gold Bio, cat. no. DTT100) Criterion XT precast gels, 4‐12% Bis‐Tris (Bio‐Rad, cat. no. 3450125) Unstained protein standard (New England Biolabs, cat. no. P7717S) SDS‐PAGE running buffer (see recipe) SDS‐PAGE staining solution (see recipe) LB agar plates (see recipe), 8 wells, with 34 μg/ml chloramphenicol, 50 μg/ml kanamycin, and 2% (w/v) glucose 10% (v/v) antifoam solution (see recipe) scFv‐H SEC running buffer (see recipe) Gel filtration standards (Bio‐Rad, cat. no. 1511901) 96‐well PCR plate (Thermo Fisher Scientific, cat. no. AB2396) 42°C heat block or thermocycler for 96‐well plate 96‐well 2‐ml deep‐well blocks (USA Scientific, cat. no. 1896‐2000), sterilized by autoclaving at 121°C for 15 min AeraSeal breathable seals (Excel Scientific, cat. no. B100) Bacterial shaking incubator at 37° and 22°C (e.g., Shel Lab S16R‐HS‐2 for small scale, New Brunswick Scientific I 26 for large scale) Spectrophotometer (e.g., Stunner, Unchained Labs) Bacterial plate incubator (e.g., Shel Lab S16R‐HS‐2) at 37°C Plate shaker (e.g., Ika MTS 2/4) at 4°C 96‐well, 0.22‐μm filter plate (Thomson, cat. no. 931919) Vacuum manifold for 96‐well filter plates (e.g., Sigma, cat. no. 57192‐U) SDS‐PAGE gel running equipment (Bio‐Rad, cat. no. 1656019) Gel imaging system (e.g., FluorChem, ProteinSimple) 250‐ml Erlenmeyer flasks (VWR, cat. no. 76531‐762) 2‐L baffled flasks (Genesis Pyrex, cat. no. 4446‐2L) 1‐L centrifuge bottles (Beckman, cat. no. C31597 ) 50‐ml centrifuge tubes (Fisher Scientific, cat. no. 14955239) Rocker 20‐ml Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 0.45‐μm syringe filters (Genesee Scientific, cat. no. 25‐242) FPLC system for protein purification (e.g., GE ÄKTA) equipped with HiLoad 16/600 Superdex 75‐pg column (Cytiva, cat. no. 28989333) 10‐kDa centrifugal concentrators (Amicon Ultra‐15, Millipore, cat. no. UFC901024) ..
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