Immunoprecipitation

Phosphoprotein Immunoprecipitation

Receptor activation by ligand addition or cross-linking, followed by immunoprecpitation of phosphoproteins, can be used it identify the signalling pathways activated by the engaged receptor. Antibody cross-linking induced signalling can be used to generate a receptor-specific signal in situations where a receptor binds to a promiscuous ligand - although care must be taken to avoid Fc-mediated signalling on cells with Fc receptors (e.g. macrophages, neutrophils). This can be achieved by the use of Fab and F(ab)₂' antibodies in place of full length antibodies. This is a detailed version of our published protocol.

Buffers & Media

Wash Buffer:

For 10ml:

• 7.99ml ddH₂O
• 20mM Tris-HCl (200ul of 1M Tris-HCl, pH 7.4)
• 0.15M NaCl (750ul of 2M)
• 2mM EDTA (40ul of 0.5M)
• 1% NP-40 (100ul)
• 10%  glycerol (1ml)
• 1mM NaVO₄ (100ul of 1M) - add immediately before use

Lysis Buffer:

To 5ml wash buffer add:

• 0.25mM PMSF (6.25ul of 200mM in ethanol)
• 200nM Okadaic acid (1ul of 1mM in DMSO)
•  10mM NaF (100ul of 500mM)
• Commercial protease and phosphatase inhibitors at the recommended concentration

5X Phenylphosphate Buffer:

Add 127mg of phenylphosphate to 1ml of PBS.

Staging

24+ Hours Prior to Experiment

1. Split/purify cells to achieve ~60-80% confluency on the day of the experiment.  For most experiments, 2-3 50mm to 100mm plates are required.
2. Culture cells are per usual.
3. Prepare all required buffers, minus protease or phosphatase inhibitors.

3-4 Hours Prior to Experiment

1. Wash plate 2X with pre-warmed serum free media.
2. Culture cells in pre-warmed serum-free media for 3 to 4 hours. This will reduce basal levels of phosphorylation generated by growth factor signalling.
3. If required, basal levels of phosphorylation can be further reduced by inhibiting focal adhesion phosphorylation after the serum-starvation step. This is done following our Inhibition of Focal Contact Contact Signalling Protocol.

Cell Stimulation & Lysis

For Mass-Spectrometry Analysis: All steps should be conducted using sterile and filtered solutions. All solutions should be kept on-ice. Gloves and other protective clothing should be worn to prevent contamination of samples from sloughed skin. Protein-free/Mass-spec reagents are prepared SDS-PAGE gels should be used. Working in a HEPA-filtered biocontainment hood may help reduce contamination.

Day 1:

1. Place samples on ice and level using a bubble level.
2. Wash plates 2X with HEPES-buffered serum-free media.
3. Remove all media and replace with 1.5ml (5cm plates) or 3ml (10cm plates) of serum-free, HEPES-buffered media with the stimuli/antibody added. Incubate 15-20 minutes, with occasional rotation of the plate to ensure even coverage.
4. Wash 3X with ice-cold PBS. If cross-linking with antibodies repeat Step 3 using the cross-linking F(ab)₂' or secondary antibody.  Wash 3X with ice-cold PBS after secondary antibody addition (if done).
5. Incubate for the desired duration. Phosphatase activity is reduced at 4^oC, so incubation of 15-20 minutes can greatly enhance phosphorylation.
6. Carefully aspirate all PBS/media from the plate.  Add 1ml of lysis buffer to the centre of each plate. Scrape cells with a cell scraper, using a new scraper for each plate.
7. Using the scraper, "squeegee" the lysis buffer to one edge of the plate and recover into a 1.5ml tube with a pipette. Pipette sample up/down 3-5 times to break up cell clumps.
8. Rotate tubes at 4^oC for 40 minutes to completely lyse the cells.
9. Centrifuge at 4^oC, maximum speed, for 15 minutes in a microfuge and recover supernatant.  Keep 50ul of each sample for controls and protein determination.
10. To each 1.5ml tube add 40ul of 4G10-conjugated agar (anti-phosphotyrosine conjugated agar). Rotate overnight (~16 hours) at 4^oC to bind phosphoproteins. Pipette tips need to be trimmed when using agarose beads to reduce retention.

Day 2:

Prepare 50ml of wash buffer (with protease and phosphatase inhibitors added), and prepare 1ml of 1X phenylphosphate (200ul of 5X into 800ul of PBS).

1. Wash 4G10-agarose 3-5X by centrifuging at 4C, max speed, in a microfuge; resuspend in 500ul of wash buffer with a glass pipette.
2. After the final wash pellet agarose and remove supernatant.  Suspend in 500ul of PBSwithout phenylphosphate. Transfer to a clean tube using a glass pipette.
3. Wash tube from steps 1-3 with an additional 500ul of PBS without phenylphospate and transfer to the new tube.
4. Centrifuge slurry for 30 seconds at maximum speed at 4^oC. Wash one more time with PBS without phenylphosphate.
5. Carefully remove all supernatant from the agarose pellet. Add 70ul of 1X phenylphosphate solution and using a clipped pipette, transfer to a clean tube.
6. Rotate at 4^oC for 30 minutes.
7. Centrifuge for 30 seconds at 4^oC, maximum speed.
8. Transfer supernatant to a clean tube, being careful to not transfer any agarose. Samples from duplicate plates can be pooled at this point.
9. Determine concentration of IPP and original sample, if desired.

SDS-PAGE & Mass Spectrometry

If performing mass-spec it is best to purchase commercially prepared, mass-spec grade gels, laemmli buffer and running buffer. This reduces the likelihood of protein contamination. For conventional immunoblotting gels and buffers prepared in-lab are sufficient. The below protocol is for mass spectrometry; for immunoblotting follow conventional immunoblotting protocols.

1. Prepare samples for SDS-PAGE by solubilizing in 4X laemmli with DTT (not beta-mercaptoethanol). Boil for 1-2 minutes to denature.
2. Run samples on a mass-spec grade SDS-PAGE gel. For best results a 4%-20% gradient gel should be used.
3. Stain gel with a mass spec grade silver stain. Down-stream processing can be simplified using a mass spectrometry grade, high--sensitivity coomassie stain.  Staining should be conducted in a hea-filtered biosafety hood to prevent contamination, using freshly cleaned staining trays/etc.
4. Carefully clean the surface of the gel scanner with a protein-removal solution.  Avoid alcohol-containing solutions as they can fix proteins onto the scanner surface. Image the gel using the cleaned scanner.
5. Using the gel image, identify bands of interest. Excise bands with a band-cutter and process as per your standard mass spectrometry protocols.