(e) Centrifuge to spin 15 mL/50 mL conical tubes at 4
C.
(f) 15 mL Conical tubes.
(g) 2 mL Microcentrifuge tubes.
(h) Small centrifuge to spin 2 mL tubes at 4
C.
(i) Vortex.
(j) Ice.
2.8 Removing Excess
Biotin from Protein
Extract
1. Amicon Ultra-4 centrifugal filter columns.
2. Protein extraction buffer (no detergents) from Subheading
2.7, step 1.
3. Centrifuge to spin conical tubes.
2.9 Affinity
Purification
1. Streptavidin-Sepharose “high performance” beads.
2. Protein extraction buffer (no detergents) from Subheading
2.7, step 1.
3. Rotator at 4
C.
4. 2% Wash buffer: 2% SDS, 20 mM Tris–HCl pH 7.5, MS grade
H 2 O.
5. 50 mM Ammonium bicarbonate (ABC) in MS grade H 2 O.
6. 1.5 and 2 mL microcentrifuge tubes autoclaved and
non-autoclaved.
7. Filter tips.
2.10 Trypsin Digest
for Mass Spectrometry
1. Trypsin from porcine pancreas.
2. 20 mM Tris–HCl pH 8.
3. 50 mM ABC.
4. 10% Stock of formic acid in glass vials (see Note 9).
5. Glass pipettes for formic acid.
6. Shaker at 37
C.
7. Speed vacuum concentrator.
3 Methods
3.1 BioID Vector
Cloning
1. Amplify PCR product of gene of interest and PCR product of
BirA* using primers designed according to the cloning kit used
(see Note 2) and gel purify PCR product.
2. Digest 25 μL of pDB (~150 ng/μL) overnight with StuI at
37
C (see Note 4).
3. Run the digested product on agarose gel and gel purify the
digested pDB band (~14.3 kb).
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Madiha Khan et al.