Coimmunoprecipitation
- Wash 30-60x 10 cm plates of asynchronously growing 293 cells (a total of approximately 6-12 x 107 cells) in phosphate-buffered saline and scrape each plate of cells into 1 ml of ice-cold EBC lysis buffer.
- Centrifuge cell lysate at 14,000g for 15 minutes at 4C.
- Pool the soluble fraction (30 ml) and add 30 ug of the anti-BHD mouse monoclonal antibody. Rock the immunoprecipitate for 1 hour at 4C.
- Add 0.9 ml of a 1:1 slurry of protein A (or protein G)-Sepharose (in 20 mM Tris, pH 8, 1 mM EDTA, 0.5% NP-40). Rock the immunoprecipitate for another 30 minutes at 4C.
- Wash the protein A or G-Sepharose four times in NETN+900 mM NaCl ad then once in NETN.
- Aspirate the protein A or G-Sepharose beads to dryness, add 800 ul of 1x Laemmli sample buffer, and boil for 4 minutes.
- Prepare the SDS-PAGE gel. The separation gel (a discontinuous 7.5-15% gel at pH 8.8) should be 20-30 cm long, the stacking gel (5% at pH6.8) should be 10 cm long, and the well itself should be 10 cm deep. Pour the stacking gel without a multiwell comb and construct the wells by inserting spacers vertically between the glass plates so that they form a well that is large enough for tha sample. Load tha sample into the well and run at 10 mA constant current overnight.
- To detect the protein bands, stain with Coomassie Blue R-250. Excise the band of interest from the gel, place it in an Eppendorf tube, and wash two times for 3 minutes in 1 ml of 50% acetonitrile. Digest the protein with trypsin while it is still in the gel, electroelute the peptides, and fractionate by narrow-born HPLC. Subject the collected peptides to automated Edman degradation sequencing on an ABI 477A or 494A machine.
Note: procedures for detection and digestion of the protein vary greatly. In particular, digestion can be performed while the protein is still in the polyacrylamide gel or after it has been transferred to a nitrocellulose or PVDF membrane. Processing of the samples for Edman sequencing or MS analysis also varies and this is best discussed with the scientist operating the machinery. A review of methods is given in Matsudaira (1993) and Link(1999).
Reagents:
Acetonitrile(50%)
EBC lysis buffer: 50 mM Tris (pH8); 120 mM NaCl; 0.5% Nonidet P-40; 5 ug/ml leupeptin; 10 ug/ml aprotinin; 50 ug/ml phenylmethylsulfonyl fluoride(PMSF); 0.2 mM sodium orthovanadate; 100 mM sodium fluoride;
20 mM Tris (pH8).
EDTA (1 mM)
1x Laemmli sample buffer
NP-40 (0.5%)
NETN: 20 mM Tris (pH8), 1 mM EDTA, 0.5% NP-40
Coomassie Blue R-250 staining:
CBB staining solution: CBB 1 g
Methanol 500 ml
Acetic Acid 100 ml
H2O to 1 liter
CBB destaining solution:
Methanol 100 ml
Acetic Acid 70 ml
H2O to 1 liter
