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Immunoprecipitation, Protein A-HRP and Background - (Dec/06/2008 )

Hi everyone,

I have a some problems with my IP, and I was wondering if someone could help me a little bit wink.gif

At the moment i´m trying to setup my IP for a protein of around ~55kDa. With normal IP protocols the heavy chain of my AB is blocking the signal from the protein, and crosslinked the primary AB doesn´t seem to work, thats why I tried to use the Exacta Cruz Protein A-HRP from Santa Cruz as a secondary (that should not detect the denatured AB).
I tried the protocol yesterday, and I had two problems:

1) I still had a signal from the heavy chain. It was weaker than with my normal protocol, but still clearly visble. Now my question: Is there still some weak interaction between the protein A-HRP and the denatured AB, or does that mean that the denaturing wasn´t 100% succsseful? I resuspend the beads in 2x sample buffer with 10% b-ME and boil them for 5 minutes at 95°C as mentioned in the protocol.

2) What blocking buffer you usually use when detecting with Protein A-HRP? The Santa Cruz protocol mentions blocking in 1xTBS + 0,05% Tween 20, which surprised me a little bit, because this buffer is usually only used for washing. I called and asked them and they said nevertheless i should follow the protocol and use it (of course) and that I never ever should use the Protein-A-HRP in TBST+5% Milk power (what i usally take for blocking).
Well, after developing my film, I had a very strong background (over the whole blot, not smear in the lanes, the blot looks like covered with sand or dust) and some unspecific bands in my total protein (where i usually have a clear band when i use primary + anti-mouse-HRP secondary).

Would be great if someone has an idea what i could change smile.gif


Hi Chekov

I have not tried Protein A-HRP, but I have a different suggestion for you:

If you are having problems with cross-linking, it can help to couple your antibody directly onto Dynabeads M-270 Epoxy from invitrogen. These magnetic beads have epoxy groups on their surface which will form covalent bonds to amine and sulphydryl groups in you antibody. The antibody will remain on the beads during elution of you IP target, so you avoid the problem of co-elution completely.