I'm looking to detect pFAK Tyr397. I've found two methods, the first is to run whole lysates on a gel, and blot for a pFAK antibody (ie pFAK-397) and the other is to IP the FAK out of the lysate, and then blot for general pTy.
Obviously the second method is better for picking up other sites of phosphorylation on FAK, but are there any other reasons for using one over the other?
It depends on your goal to achieve. Both methods might work. But it depends on the validation and experimental confirmation of the functional quality (specificity and affinity) of the antibody you are going to use for IP. If this is the same Ab (pFAK-397) you need to make sure that this Ab will precipitate your protein and will not precipitate any other proteins with phospho-Tyr. And the same is true, if you decide to use this Ab for WB of whole cell extract (will bind only your protein and no any other Tyr-phosphorylated proteins). If you 100% sure that the Ab is great (confirmed in experiments) I would recommend to use first IP method. If your goal is to determine the relative content of your protein phosphorylated at Tyr397 I would recommend to run WB on whole cell lysates.
In general, immunoprecipitation is more expensive and time consuming experiment than just running whole cell extracts on western blot. The problem is that some antibodies have difficulties to detect proteins in whole cell extracts (or they stain lot of unspecific proteins too, so interpretation can be confusing), which is why people immunoprecipitate the protein and then use either general pTyr antibody or antibody specific to a particular protein (in your case pFAK-397) to detect phosphorylation. If I were you, I would try the pFAK-397 on whole cell extracts first. If it works, great. You can even use it to quantify differences in phosphorylation under different experimental conditions. If it does not, than try to IP FAK first and detect phosphorylation by western blot.
If i Use IP FAK, would I then use FAK/pFAk for detection, or pTyr for detection? Many people seem to blot for total phospho-tyrosine after FAK IP...
Thanks for your answers!
Depends on what are you trying to see. General anti-pTyr Ab is going to detect everything phosphorylated on Tyr in your sample. So if a protein gets co-isolated with FAK during your IP, then you could detect its phosphorylation too (additional band on WB). If FAK has more Tyr besides Y397, then you could also detect phosphorylation on these other residues meaning you can't conclude that FAK gets phosphorylated on Tyr397. You can only say that it gets phosphorylated. If you specifically want to look at phosphorylation at Y397 (if this residue is somehow relevant for the signaling pathway you are studying, e.g. Y397 is phosphorylated only when FAK is activated), then you need to use the pY397 specific antibody.
Oh, I should also clarify that sometimes the general anti-pTyr antibodies do not detect all pTyr residues. It is rare, but it did happen in our lab.
Another advantage of an IP/IB experiment is the ability to IP from a larger amount of protein than you would ever be able to load/run on a gel. Thus, you may be able to examine phosphorylation of much lower copy number targets. You should probably IP with the antibody to the total protein then probe with the anti-phospho protein. Finally, strip the blot and reprobe for total FAK. It would be better if the reprove was with a different antibody than the one used in the original IP. You can also cross-link the antibody in the IP to the beads to 'clean up' the final IB, if needed.
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