Wester VS Immunoprecipitation -Da Paradigm...?!?! - Western Blot & Immunoprecipitaion & Ab (Dec/06/2003 )
This is a verrrry nasty situation that I just don't understand...
...Maybe some of you would have a good explanation........please...
I'm doing the IP of the TCR zeta chain, and with the same Ab that
I use for the IP I'm doing the WB and I obtain a good specific signal.
However, I just see the basal isoform of the zeta chain (16kDa) and I don't see all the isoforms (21 and 23 kDa) that are phosphospecies
of the zeta chain. (T cells have been activated, etc...)
Indeed, I only see those phosphorylated isoform when I'm doing a
WB with an Ab against phosphotyrosine.
Why I can't see all the isoforms of the zeta chain with the Ab that
I use to immunoprecipitate ALL those isoforms........????
This situation is also prensent in all paper thats study the TCR
phosphorylation, but no one is talking about this...!?!?
Hope that I have been clear in my explanations...
--Please--- Help me to resolve this paradigm
Thanks in advance!
Some ideas that may help you sleep.
1. Maybe the epitope site is blocked by the p-Tyr moiety/moieties. There is a pretty big shift in molecular size (16 to 21/23kDa!) so you might imagine that all the phosphates could sterically get in the way of antibody binding. Check the epitope to which the antibody was designed for tyrosines. The negative charge of the phosphate moieties could also change the pI of the protein affecting its transfer to membranes at a certain transfer buffer pH. Maybe check a commassie of the gel before transfer to see if all isoforms are indeed IPed.
2. Maybe the phosphoproteins are there but not as abundant. Phosphorylation is not all or none. What if 5-10% of your target protein is phosphorylated at any given time? The protein level may serve to modulate the sensitivity of a response whereas activation a small percentage of the protein pool may be enough for signal transduction.
the best idea will be to use a seperate ab for IP and a different ab for WB you can also use ab raised in different species for IP and WB.
when you are using phospho ab's you can only see phosphoproteins you cannot see non phosphoproteins
i would recomend victors suggestions as well.
You are not seeing the isoforms because the titre level needed to detect those species is higher than the parent isoform. Remember that immunoprecipitation is a liquid phase reaction; it is more efficient while western blot is semi-solid phase; the targets are immobilized while a liquid phase antibody is searching for something to bind to. We recommend that you donot change the titre but increase the incubation time with first antibody to 36 hours at 18C. The isoforms will surprise you by showing up this time!
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