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getting rid of non specific bands in IP experiments - (Mar/21/2012 )

Hi,
I have been having some trouble lately with non specific bands showing up when I IP my samples. Regardless of the anitobody that I have been using ( V5, HA) both of which are antimouse antibodies, I have seen a non specific band around ~ 60KD and ~25-30KD in my IP samples. But not in my Input. If these are the heavy and light chains in the IgG antibody what is best way to get rid of these non specific bands?
It is interfering with my experiment and with different antibodies I still have not been able to get rid of it. I have been using the sepharose A beads for the reactions. Any suggestions would be appreciated.

-molecule-

Do you know that you need to use antibodies from different species for your IP and WB? for example if you use an antibody raised in rabbit for IP and an antibody raised in mouse for your WB you will not see the heavy and light chain anymore. your problem is that your secondary antibody during WB lables your heavy and light chains of your IP antibody. this is a typical problem in IP.

-Curtis-

I have also seen the protein (G in my case) eluting from the beads. This is around 30 kDa, as determined by running a beads with no antibody control.

-bob1-

bob1 on Thu Mar 22 19:55:15 2012 said:


I have also seen the protein (G in my case) eluting from the beads. This is around 30 kDa, as determined by running a beads with no antibody control.

good to learn that. I always thought the beads ARE the protein G or A. never realized they coat the beads with them. I never faced this issue however. I ran beads with no antibody as control many times.

-Curtis-

Usually beads are sepharose (agarose) or acrylamide with conjugated protein G or A. You can buy pure protein G and A if you want - it used to be used for IPs, but the beads are much more efficient, as the formation of precipitable and pelletable complexes just with the protein G/A and Ig's of some sort was difficult

-bob1-

thank you for the information. I actually tried using different antibodies for the IP and the WB. ( Ie: used a rabbit antibody for the IP and a mouse for the WB) but was still having the "non specific band problem" but the intensity was a bit less. Also I;m trouble shooting the amount of antibody to use for the IP process, and with the increase of antibody I was seen an increase in the intensity of this band as well.
I'm concerned if there is anything incorrect in the process of the IP that I'm doing given that I can't pull down anything and if the protein is getting degraded in the process. Any help/suggestions would be greatly appreciated!

-molecule-

HI,

thank you for the suggestions. and it was helpful in solving my problem. Right now I'm faced with a different problem of being unable to perform the Co-IP. The IP experiments have been working ( so far) and I have been able to determine the concentration of antibody that I need to use etc, but the positive control for my Co-IP is not working. These are two proteins that are known well to interact with one another.
Since its HA tagged I have tried different antibodies etc but nothing has been working so far. I see a signal in the Input lane but not in my CO-IP samples.
If anyone has any suggestions that will be helpful..

-molecule-

Is the tag near the domain(s) that interact? - if so, it may be affecting the interaction, hence no co-IP.

-bob1-