4. NuPAGE
® Pre-Cast Gel System, 4–12% Bis/Tris Gel.
5. NuPAGE
® LDS Sample Buffer (Thermo Fisher Scientific).
6. Refrigerated tabletop centrifuge.
7. Shaker/roller.
8. Speed vacuum concentrator.
9. Protein quantification kit.
10. Imperial protein stain (Thermo Fisher Scientific).
11. Silver stain kit (Thermo Fisher Scientific).
12. Spectrophotometer.
13. Mass spectrometer: Q Exactive Plus Hybrid QuadrupoleOrbitrap online with a nanoLC Ultimate 3000 chromatography system (Thermo Fisher Scientific™, San Jose, CA).
2.2 Basic Reagents
for Immunopurification
1. Puromycin dihydrochloride antibiotic.
2. Poly(ethyleneimine) solution, Transfection Reagent.
3. pIRES puro 3 vector (Clontech).
4. ANTI-FLAG
® M2 Affinity Gel (Sigma-Aldrich).
5. Lysis buffer: 10% (v/v) glycerol, 50mM HEPES–NaOH pH
8.0, 150mM NaCl, 2mM EDTA, 0.1% (v/v) IGEPAL
CA-630, 2mM DTT, protease inhibitor cocktail (SigmaAldrich), and phosphatase inhibitor cocktail (Sigma-Aldrich).
6. Specific antibodies against the protein of interest and known
interacting proteins.
7. Monoclonal ANTI-FLAG
® M2 antibody produced in mouse
(Sigma-Aldrich).
8. NuPAGE
TM LDS 2X Sample Buffer (Thermo Fisher
Scientific).
9. 3x FLAG
® peptide (Sigma-Aldrich).
2.3 Basic Reagents
for Peptide-Based
Purification
1. NHS (N-hydroxysuccinimide) activated Sepharose 4 Fast Flow
(GE Healthcare Life Sciences).
2. Isopropanol.
3. 100 mM MES (2-(N-morpholino)ethanesulfonic acid sodium
salt), pH 6.8.
4. 1 mM HCl.
5. 200 mM ethanolamine, pH 8.
6. 100 mM Tris–HCl pH 8.
7. 100 mM Sodium acetate pH 4.
8. Preservative solution: PBS with 0.2% azide.
9. Synthetic peptides.
20
Avais M. Daulat et al.
® Pre-Cast Gel System, 4–12% Bis/Tris Gel.
5. NuPAGE
® LDS Sample Buffer (Thermo Fisher Scientific).
6. Refrigerated tabletop centrifuge.
7. Shaker/roller.
8. Speed vacuum concentrator.
9. Protein quantification kit.
10. Imperial protein stain (Thermo Fisher Scientific).
11. Silver stain kit (Thermo Fisher Scientific).
12. Spectrophotometer.
13. Mass spectrometer: Q Exactive Plus Hybrid QuadrupoleOrbitrap online with a nanoLC Ultimate 3000 chromatography system (Thermo Fisher Scientific™, San Jose, CA).
2.2 Basic Reagents
for Immunopurification
1. Puromycin dihydrochloride antibiotic.
2. Poly(ethyleneimine) solution, Transfection Reagent.
3. pIRES puro 3 vector (Clontech).
4. ANTI-FLAG
® M2 Affinity Gel (Sigma-Aldrich).
5. Lysis buffer: 10% (v/v) glycerol, 50mM HEPES–NaOH pH
8.0, 150mM NaCl, 2mM EDTA, 0.1% (v/v) IGEPAL
CA-630, 2mM DTT, protease inhibitor cocktail (SigmaAldrich), and phosphatase inhibitor cocktail (Sigma-Aldrich).
6. Specific antibodies against the protein of interest and known
interacting proteins.
7. Monoclonal ANTI-FLAG
® M2 antibody produced in mouse
(Sigma-Aldrich).
8. NuPAGE
TM LDS 2X Sample Buffer (Thermo Fisher
Scientific).
9. 3x FLAG
® peptide (Sigma-Aldrich).
2.3 Basic Reagents
for Peptide-Based
Purification
1. NHS (N-hydroxysuccinimide) activated Sepharose 4 Fast Flow
(GE Healthcare Life Sciences).
2. Isopropanol.
3. 100 mM MES (2-(N-morpholino)ethanesulfonic acid sodium
salt), pH 6.8.
4. 1 mM HCl.
5. 200 mM ethanolamine, pH 8.
6. 100 mM Tris–HCl pH 8.
7. 100 mM Sodium acetate pH 4.
8. Preservative solution: PBS with 0.2% azide.
9. Synthetic peptides.
20
Avais M. Daulat et al.