Expressing:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
Plasmid Preparation:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
Purification:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
Modification:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
Cell Culture:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
Transfection:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
Saline:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
Lysis:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
Protease Inhibitor:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
Immunofluorescence:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
Staining:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
Electron Microscopy:Article Title: Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.
Article Snippet: .. Materials Expression plasmid for bait protein of interest AirID gene (Addgene plasmid #182210; also see Supplemental Files in Supporting Information) DNA plasmid purification kit of choice (e.g., PureYield Plasmid Miniprep System, Promega, cat. no. A1222; ZymoPURETM II Plasmid Purification Kit, midiprep, Zymo Research, cat. no. D4201) HEK 293T cells (or other appropriate cell line) 1× Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (or other appropriate cell culture medium) jetPRIME transfection reagent and buffer (PolyPlus, cat. no. 101000015) 100 mM biotin (see recipe) 1× Dulbecco’s phosphate-buffered saline (DPBS) (Corning, cat. no. 21-031-CV) Co-immunoprecipitation (IP) lysis buffer (see recipe) or other lysis buffer Protease inhibitor cocktail (Millipore, cat. no. P2714-1BTL) Acetone, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 10000) Schaack, Sullivan and Mehle 3 of 26 Current Protocols 26911299, 2023, 3, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.702 by C ochrane Israel, W iley O nline L ibrary on [06/08/2023]. .. 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 Methyl alcohol, high grade, suitable for immunofluorescence staining (Electron Microscopy Sciences, cat. no. 18510) 1%-3% (w/v) bovine serum albumin (BSA) in DPBS Fluorophore-conjugated streptavidin, Alexa Fluor 488 conjugate (Invitrogen, cat. no. S11223) Fluorophore-conjugated antibodies to detect bait and AirID fusion proteins (e.g., human anti-V5 epitope tag Alexa Fluor 594, Novus, cat. no. NBP2-81035AF594) Mounting medium containing DAPI stain (Vectashield, H-1200) 6-well tissue culture plates Microplate sealing tape Platform rocker Microcentrifuge tubes Microcentrifuge Glass coverslips coated with fibronectin over a PDL layer (Neuvitro, cat. no. GG-12-1.5-Fibronectin) Glass microscope slides Needle Forceps Nail polish Generate plasmid encoding AirID fused to bait protein The AirID amino acid and gene sequences are available from Kido et al. (Kido et al., 2020).
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