Many of the protein-protein interactions present in intact cells are maintained when cells are lysed under nondenaturing conditions. This rhopper can be used to detect and characterize protein-protein interactions that are relevant under physiological conditions. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Determination of binding protein assay by immunoprecipitation
Materials and Instruments
Suitable cell lines grown in the medium Move makings For more product details, please visit Aladdin Scientific website.
Acetonitrile Kaumas Brilliant Blue R-250 EBC Lysis Buffer NETN Phosphate Buffer Solution SDS Gel Spiking Buffer Trifluoroacetic Acid Trypsin Trypsin Digestion Buffer Antibodies to Precipitate Target Proteins Control Antibodies Discontinuous SDS-PAGE Gradient Gel
Boiling Water Bath Protein A-Sepharose Plate Shaker
Buffers and solutions
Dilute storage solution to appropriate concentration
Acetonitrile (50%)
Caulmers Brilliant Blue R-250
EBC Lysis Buffer
50 mmol/L Tris-Cl (pH 8.0)
120 mmol/L NaCl
0.5% (V/V) Nonidet P-40
5ug/ml white gastrin
10ug/ml Peptidase
50ug/ml PMSF
0.2 mmol/L orthovanadate
100 mmol/L NaCl
NETN
20 mmol/L Tris-Cl (pH 8.0)
1 mmol/L EDTA
100 mmol/L NaCl
0.5% Nonidet P-40
NETN, containing 900 mmol/L NaCl
phosphate buffer solution
1xSDS Gel Spiking Buffer
Trifluoroacetic acid (TFA) (0.1%, m/V, sequencing grade)
Enzyme and Buffer
Trypsin
250ug/ml of bovine trypsin was prepared in 200 mmol/L ammonium bicarbonate (pH 8.9) (sequencing grade, Boehringer Mannheim) solution
Trypsin Digestion Buffer
0.02% (V/V) Tween-20
200 mmol/L ammonium bicarbonate (pH 8.9)
Antibody
Antibodies to precipitated target proteins
Control Antibodies
Gel
Discontinuous SDS-PAGE Gradient Gel
Separation gels (7.5%-15% discontinuous gels at pH 8.8) should be 20-30 cm long, concentration gels (5% gels at pH 6.8) should be 10 cm long, and the wells themselves should be 1-2 cm deep (the percent concentration of the gels will vary with the target protein). Instead of using a porous comb to pour the gel concentrate, the wells are made by inserting spacers vertically between the glass plates, which creates a sufficiently large well. See Appendix 8 for details on SDS-PAGE.
Specialized Equipment
Boiling water bath
Protein A-Sepharose
Preparation of Protein A-Sepharose in NETN buffer for 1:1 suspension
Plate shaker
Additional reagents
Steps 10 and 11 of this protocol require reagents and instrumentation for peptide profiling, peptide purification, and sequencing (see Chapters 62 and 63 of Spector et al. 1998).
Cells and Tissues
Suitable cell lines grown in culture medium
Methods
Important: This protocol is used to identify pVHL binding proteins. Conditions should be optimized for the target protein.
1. Wash 30 10 cm culture plates with phosphate buffer with suitable cells (approx. 6X107 cells in total). Scrape the cells from each plate into 1 ml of ice-cold EBC lysis buffer.
2. Transfer each ml of cell suspension to a microcentrifuge tube and centrifuge at 4°C for 15 min at maximum speed in a microcentrifuge.
3. Collect the supernatant (approx. 30 ml) and add 30ug of appropriate antibody and shake the immunoprecipitate at 4°C for 1h.
4. Add 0.9 ml of Protein A-Sepharose suspension and shake the immunoprecipitate at 4°C for a further 30 min.
5. Wash the Protein A-Sepharose mixture with NETN containing 0.9 mmol/L NaCl and repeat the wash 5 times. Finally, wash once with NETN.
6. Aspirate the liquid portion of the mixture. Add 800ul of 1XSDS Gum Spiking Buffer to the beads and boil for 4 min.
7. Add the sample to a large well of discontinuous SDS-PAGE gradient gel and electrophoresis at a constant current of 10mA overnight.
8. Observe the protein bands by staining with Caulobacter blue (Appendix 8).
9. Cut the target bands from the gel, put them into a microcentrifuge tube and wash them twice with 1ml 50% acetonitrile for 3 min each time.
10. Digest the proteins in the gel with trypsin and then electroelute the peptides.
The peptide is then electroeluted. a. Transfer the gel to a clean surface and allow it to not dry completely.
b. Add 5ul of Trypsin Digestion Buffer and 2ul of Trypsin Solution.
c. After the gum adsorbs the trypsin solution, add 5ul of trypsin buffer at a time until the gum strip returns to its original size.
d. Place the gel in a microcentrifuge tube, soak in Trypsin Digestion Buffer and incubate at 30°C for 4 h. Terminate the reaction by adding 1.5ul of 0.1% trifluoroacetic acid.
The observation of the proteins and the digestion steps were highly variable. In fact, proteins can be digested while they are still in the polyacrylamide gel or after they have been transferred to nitrocellulose or PVDF pancreas.
11. Separation of peptides by narrow-well HPLC. The collected peptides are subjected to automated Edman degradation sequencing on an ABI477A or 494A machine.
Sample handling for sequencing or Edman analysis varies and is best discussed with the person operating the instrument. For a review of the method, see Matsudaira (1993), Link (1999), and Spector et al. (1998, Chapters 62 and 63).