uneven bands from Immunoprecipitation - (Nov/06/2007 )

I am interested in the level of phosphorylation of protein X in response to drug treatment. There are multiple isoforms of protein X, I am interested in isoform Xa. There are no commercially avaliable antibodies for phosphorylated Xa. Therefore, I am immunoprecipitating for Xa (using Protein A agarose beads) and then western bloting for phosphorylated X (so in theory my band will represent phosphorylated Xa). I am using antibodies raised in the same species, so on my western I also see bands for the heavy and light chains of my antibody (they do not run at the same size as my protein of interest so this is not a problem). Even when I IP for Xa and blot for Xa (a protocol that should not see differences in Xa expression levels in response to drug treatment) I am getting different sized heavy and light chain bands with my current method (in addition to different sized Xa bands). Because I am getting different sized anitbody bands, even when looking at a static protein, this leads me to believe that I have a problem in the IP (maybe not a uniform amount of beads in each sample) and not that I am actually seeing a treatment effect. Dose anyone have any ideas of what might be going on/suggestions on how to fix this problem?

Thanks for the help in advance....

P.S. I have attached a picture of IP Xa, IB Xa so you can see the different band sizes.

[attachment=3798:IP_a_IB_a.jpg]

P.P.S. I am not sure how well I explained my problem so please write back with any questions for clarification.

Ummm, if I understand correctly, the bright black band in the photo is the
IgG Heavy Chain right?
If so, this heavy chain is the same size in ALL the lanes.
The minor variation is due to slight problems with gel running. The heavy chains are a little different due to minor differences in migration. That's all.
The faint bands above the heavy chain are also all the same size.

Thanks for your reply, but if you enlarge the picture the bands are definitely not all the same size (for example the first band is way thicker than the next few). And yes the bright band is the IgG heavy chain.

Hi again,

So what you mean to say is the the bands are not of equal intensity, not that
they are of a different size. A different size would mean a difference in kDa.
The IgGs are of approximate equal intensity. There may be some variation in your
pull down with the beads. This may be from suctioning off some beads during the washes.
Overall they are okay I think.
The larger concern is that the protein you are looking at is of very low intensity.
I would focus on improving the signal from the immunoprecipitated protein first.
Does this protein give a signal on Western? If so the signal should be significantly stronger
on an IP.

You are correct, what I mean to say is that the bands are different intensity. The protein actually gives a much stronger on a western than with the IP.

Thanks for the advice.

You can't compare the Ig bands, because they are overexpressed. final point.

If we check your band of interest, yes you have a problem.
When you wash the beads, be careful when you discard the supernatant after centrifugation : let some buffer over the beads (around 1 mm height), wash three times, the last time, use flat loading tips to collect all supernatant. be careful, because some beads will stick to the tip (that's why you should do it only once, the last time).
good luck