I am finding which domain of my protein A interacts with protein B. I have designed a 250kDa protein A into three fragment tagged with HA at both of their terminal. I have tried to pull down the Protein B to check which domain of Protein A interacts with it. But I find the consequetive presence of all the bands in pre-immune as well as pull down proteins.
Troubleshooting: I tried earlier using 1% NP-40 Buffer so I changed it to 1% Triton X-100 buffer. Both gives similar result but when I used strong buffer like RIPA I can't find any interacting band or pre-immune.
Also, I have used other pre immune anti sera but didn't affected any result.
What should I do next or if you feel any other possibility please suggest.
First of all, cutting a large protein in three portions, tag each individual with HA, and then hope that each portion folds up the same way as if it were part of the whole protein is not a good strategy, unless it is well-established that the protein has three independent domains that fold up independently of each other.
Secondly, when I test if two proteins interact with each other, I will do several controls. I express each of the two proteins alone (two controls) and together, and test by simple western blotting if expression is reproducible. Then I will do the IP on each of the three samples with each of the two antibodies and then test with the second antibody. Controls with pre-immune serum are good, but you should also do a control where you do an IP without any serum, just add protein A sepharose or protein A magnetic beads. Another control is to do an IP with antibodies, but leave the protein sample out. If you still see something in the western, then you are detecting the primary antibody or protein A released from boiling the pellet. This is not a problem when you do in vivo labelling and do autoradiograms after IP and SDS-PAGE, because neither the antibodies nor the protein A are radioactive, but few people do this nowadays and instead do everything with antibodies. But sometimes people forget that testing an IP with a western blot means that the two antibodies have to be raised in different animals, otherwise the western will pick up the first antibody heavy chain because it is released by boiling from protein A. Some protein A also gets released by boiling.
Last but not least, to get specific IPs and avoid aggregates, I usually do a high speed spin after incubating the protein extract with antibody, transfer the supernatant to a fresh tube to avoid unspecific protein aggregates, and then add protein A sepharose or protein A magnetic beads. Protein sepharose sediments without centrifugation just by leaving the tubes on ice for 5 minutes, and this avoids pelleting any residual protein aggregates.
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