10. Bradford reagent.
11. 25G needles.
12. 1-mL syringes.
13. Rotating wheel for 1.5-mL tubes.
2.5
SDS-Polyacrylamide
Gel Electrophoresis
and Western Blotting
1. Lysis buffer I: 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM
EDTA, 0.5% NP-40, 0.5 mM DTT, protease inhibitor cocktail.
2. Stacking gel buffer: 1 M Tris–HCl, pH 6.8.
3. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8.
4. Thirty percent acrylamide/bisacrylamide solution (37.5:1).
5. Running buffer: 25 mM Tris–HCl, 192 mM glycine,
0.1% SDS.
6. Sample loading dye (5Â): 250 mM Tris–HCl, pH 6.8, 5%
β-mercaptoethanol, 0.02% bromophenol blue, 10% SDS, 30%
glycerol.
7. Low-fluorescence PVDF membranes (see Note 8).
8. Trans-Blot Turbo Transfer System and reagents (see Note 9).
9. Infrared dye-conjugated streptavidin (see Note 8). Store at
4
C.
10. Precast protein gels and reagents (see Note 10).
11. Coomassie staining solution: 0.02% (w/v) Coomassie blue
stain G-250, 5% (w/v) aluminum sulfate-(14–18)-hydrate,
10% (v/v) ethanol (96%), 2% (v/v) orthophosphoric acid
(85%). Store at room temperature.
12. Destaining solution: 10% (v/v) ethanol (96%), 2% (v/v) orthophosphoric acid (85%).
13. Sterile 15-cm bacterial dishes.
14. Sterile scalpels.
3 Methods
3.1 Cloning of Two
Genes of Interests
in Split-BioID Plasmid
1. Design primers to amplify the two genes to be tested, so that
restriction sites are included that allow subcloning in splitBioID plasmids (see Note 11).
2. Perform PCR to amplify both genes, run the reactions on a 1%
agarose-TAE gel and cut slices of agarose gel that encompass
the amplified DNA. Extract and purify the amplified DNA
using a standard DNA gel extraction kit.
3. Digest amplified DNA and acceptor plasmid(s) with the relevant restriction enzymes. Typically 2 μg of plasmid is mixed
with 1 μL of each restriction enzyme in a total volume reaction
of 20 μL, whereas 25 μL of the purified PCR products is mixed
Context-Specific Proximity-Dependent Proteomics with Split-BioID
307
11. 25G needles.
12. 1-mL syringes.
13. Rotating wheel for 1.5-mL tubes.
2.5
SDS-Polyacrylamide
Gel Electrophoresis
and Western Blotting
1. Lysis buffer I: 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM
EDTA, 0.5% NP-40, 0.5 mM DTT, protease inhibitor cocktail.
2. Stacking gel buffer: 1 M Tris–HCl, pH 6.8.
3. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8.
4. Thirty percent acrylamide/bisacrylamide solution (37.5:1).
5. Running buffer: 25 mM Tris–HCl, 192 mM glycine,
0.1% SDS.
6. Sample loading dye (5Â): 250 mM Tris–HCl, pH 6.8, 5%
β-mercaptoethanol, 0.02% bromophenol blue, 10% SDS, 30%
glycerol.
7. Low-fluorescence PVDF membranes (see Note 8).
8. Trans-Blot Turbo Transfer System and reagents (see Note 9).
9. Infrared dye-conjugated streptavidin (see Note 8). Store at
4
C.
10. Precast protein gels and reagents (see Note 10).
11. Coomassie staining solution: 0.02% (w/v) Coomassie blue
stain G-250, 5% (w/v) aluminum sulfate-(14–18)-hydrate,
10% (v/v) ethanol (96%), 2% (v/v) orthophosphoric acid
(85%). Store at room temperature.
12. Destaining solution: 10% (v/v) ethanol (96%), 2% (v/v) orthophosphoric acid (85%).
13. Sterile 15-cm bacterial dishes.
14. Sterile scalpels.
3 Methods
3.1 Cloning of Two
Genes of Interests
in Split-BioID Plasmid
1. Design primers to amplify the two genes to be tested, so that
restriction sites are included that allow subcloning in splitBioID plasmids (see Note 11).
2. Perform PCR to amplify both genes, run the reactions on a 1%
agarose-TAE gel and cut slices of agarose gel that encompass
the amplified DNA. Extract and purify the amplified DNA
using a standard DNA gel extraction kit.
3. Digest amplified DNA and acceptor plasmid(s) with the relevant restriction enzymes. Typically 2 μg of plasmid is mixed
with 1 μL of each restriction enzyme in a total volume reaction
of 20 μL, whereas 25 μL of the purified PCR products is mixed
Context-Specific Proximity-Dependent Proteomics with Split-BioID
307