Hi,
I am troubleshooting a protocol to cross-link antibodies to beads. I
am using a mouse monoclonal antibody and Gammabind protein G
sepharose beads. I am following what seems to be a standard protocol
using DMP to cross-link the antibody to the beads.
To check the efficiency of the cross-linking, I run aliquots taken at
various stages of the protocol on SDS-PAGE, followed by Coomassie
staining. According to this gel, most of my antibody remained in the
supernatant and was NOT bound to the beads. Additionally, the cross-
linking seemed inefficient because some heavy chain and light chain
could be seen in the lane containing cross-linked antibody + beads.
However, when I used these very same antibody-conjugated beads for
immunoprecipitation, removal of the target protein was almost
complete (>95%), and the antibody-conjugated beads worked better than
antibody that was uncoupled to beads. Beads alone, without antibody,
did not pull down any of the target protein. The western blot was
done using a rabbit polyclonal antibody to avoid possible artifacts.
I am suspicious of this result because it does not make sense, given
the lack of efficiency of the cross-linking. Therefore, I would like
to ask for your help with the following:
1. What could explain the unexpected success of the IP, given the
poor binding of antibody to the beads?
2. Does anyone have suggestions for improving the efficiency of the
crosslinking?
3. In the next round I will try BSA as a negative control for cross-
linking, to see if BSA-conjugated beads also pull down my target
protein. Are there any other controls that I should consider?
Many thanks in advance for your help,
Irit Rappley
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