I am doing Immunoprecipitation experiment with A/G beads for pulling down my protein of interest. Even though playing around with IgG rabbit antibody concentration to the protein lysate, still I am unable to avoid IgG bands. The whole lane is getting saturated and I am really worried how should I be avoiding this? The attached picture will give you an idea and kindly suggest me ways to avoid this IgG band. I am sure that the IgG lane is getting over saturated and hence whole lane is dark. While when I tried to adjust conytrast etc using Image lab, my IP bands of treatments are not seen. Kindly help.
I use 1ul antibody for pull down, and now decreasing rabbit IgG to 0.2ul, but still it doesnt help.
Thanks a lot in advance!
I presume that you are pulling down the target antigen in the lysates using the antibody bound to protein A/G beads. Then running that on gel/ immunoblot. If this is the case then you will also be eluting the IgG antibody which is messing up your profile. What you need to do first is to covalently couple the antibody to the protein A/G beads so that only your antigen is released from the beads. The company New England Biolab (www.neb.com) has a very nice protocol to carry out this process using magnetic protein A/G beads. But you can use the protocol using non-magnetic beads but replace using a magnet for purification with simple centrifugation.
If not allready tryed i will suggest first to remove/leave behind IgG by
simpel ammonium sulfat
gradients
Best wishes
Hello Peter, Can you please elaborate the procedure that you were suggesting?
Thank you!
It is not clear for me in detail what you are doing, and why. Therefore I can not help.
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