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So class, I have a quick question about Co-IP detection. In the case of MULE, my antibody would bind EGFR (bound to MULE), then wash out unbound crap to have Ab-EGFR-MULE. Under normal circumstances, I would just run this on a gel and do a western blot against MULE to confirm binding, but without the MULE Ab this doesn't work. Is there another way of detecting secondary targets in Co-IP? otherwise I'm just thinking of doing a different binding assay, but I'm curious about how the MULE discoverers did it.

Also, someone talked about using RT-PCR from MULE RNA to isolate the gene. But given the similarities with RAS, wouldn't we end up with a mixture of the two? I'm thinking in terms of primers, because the only differences are in UTRs, which you won't have in the cDNA... so how can the primers be specific for just MULE?

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  1. Hi Grace! We don't have MULE Abs yet, but some RAS Abs recognize MULE as well… you could use one of these RAS Abs, unless you are expecting that RAS may also come out during the Co-IP. Is there some way to ensure that you won't get RAS in your Co-IP with EGFR? You could also try tagging the MULE gene (His, FLAG, etc) and purify on a column, and then run and blot that using EGFR Abs to see if EGFR is bound to MULE. I'm not sure if any of that helps/ if any of that is even valid.

    With regards to the RT-PCR, the primers would be specific for the UTRs of the MULE mRNA transcript, which will then be converted into a cDNA PCR product via RT-PCR. In this case, this cDNA would actually end up containing sequences that encoded for the UTRs because they were used primers, but we could perform further PCR on the cDNA obtained to select for just the MULE gene; at least, that's what I was hoping would happen. 

    1. Grace Uwase AUTHOR

      Yeah I thought about tagging MULE but I was trying to avoid doing more work, so I'm considering other binding assays. I asked about the RT-PCR because I had already proposed using PCR to isolate DNA directly because then I would have access to the UTRs, but then you talked about RT-PCR and I was considering what the advantage would be. If either way we end up with UTRs in our isolated gene, I'm not sure if it's worth the reverse transcriptase.

      So I apparently forgot to hit "post" so this was just hanging out unposted lol