Hi,
I am trying to do co-IP with two different proteins, one tagged with GFP and the other with HA. My input shows that both the genes are expressed but when I perform the GFP-pulldown I can only see the GFP tag and no protein (based on the size) with the GFP antibody and don't see the HA tagged protein at all.
Has anyone faced this issue? And what could be the possible reason?
I am using Chrometek GFP pulldown-magnetic beads.
Thanks in advance.
Hi Sapinder, a lot of things could be happening, do you have any more information you could add? Perhaps your GFP tag is cleavable (what kind of vector do you have the gene in?) or the proteins might not be binding to the beads/each other and could be in the wash fraction?
Hi Morgan Marshall,
Sorry for late reply. My GFP-tagged protein is in c-terminal eGFP vector under 35S promoter. We know that the two proteins interact but I keep seeing a lot of cleaved eGFP which results in very low signal of the protein as such. I have decided to switch to His-tag pulldown, as you said may be my eGFP keeps cleaving. Just have to clone everything again :-(
Thank you!!
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