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== 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<ref name="Heit et al, Dev Cell, 2013 Feb 25;24(4):372-83">http://www.ncbi.nlm.nih.gov/pubmed/23395392</ref>.

 

== 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 ===

#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.
#Culture cells are per usual.
#Prepare all required buffers, minus protease or phosphatase inhibitors.

 

=== 3-4 Hours Prior to Experiment ===

#Wash plate 2X with pre-warmed serum free media.
#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.
#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 [[Inhibition of Focal Contact Signaling|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:'''

#Place samples on ice and level using a bubble level.
#Wash plates 2X with HEPES-buffered serum-free media.
#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.
#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).
#Incubate for the desired duration. Phosphatase activity is reduced at 4oC, so incubation of 15-20 minutes can greatly enhance phosphorylation.
#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.
#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.
#Rotate tubes at 4oC for 40 minutes to completely lyse the cells.
#Centrifuge at 4oC, maximum speed, for 15 minutes in a microfuge and recover supernatant.  Keep 50ul of each sample for controls and protein determination.
#To each 1.5ml tube add 40ul of 4G10-conjugated agar (anti-phosphotyrosine conjugated agar). Rotate overnight (~16 hours) at 4oC 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).

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

#Prepare samples for SDS-PAGE by solubilizing in 4X laemmli with DTT (not beta-mercaptoethanol). Boil for 1-2 minutes to denature.
#Run samples on a mass-spec grade SDS-PAGE gel. For best results a 4%-20% gradient gel should be used.
#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.
#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.
#Using the gel image, identify bands of interest. Excise bands with a band-cutter and process as per your standard mass spectrometry protocols.

 

== References ==

<references />

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