I did some HEK 293 cell, plasmid transfections to purify my protein of interest which was Flag tagged at N-terminus. Along with this I also transfected a Flag positive control plasmid separately. Isolated the protein using RIPA buffer for western blot. Did a SDS-PAGE 4% stacking/ 8% running gel. Transferred to PVDF membrane 40V, 8hrs in ice, inside a cold room. The transfer buffer has 20% methanol and 0.05% SDS.
Incubated with each respective antibody over night at 4C. After immunoblotting I could detect my protein of interest using the specific antibodies as expected, but when I used Anti-Flag antibody I could not see a band for my protein of interest, but I could detect my positive control for flag.
I was thinking may be if my protein was re-natured/ or assumed a different conformation during transfer conditions which sterically hinder the flag tag accessing with its antibody. How should I solve this problem?
Any comments or suggestions are much appreciated.
One possibility is that the FLAG tag was lost from the N-terminus due to proteolysis. To test this, use the anti-FLAG antibody to see if you can immunoprecipitate your protein.
Thank you for your comments.
@ Arvinth: My flag tagged control was only for anti-flag antibody and not for my protein of interest (POI). My POI has a different antibody to detect and doesn't work with flag tagged positive control. I think I need to redo the transfection in-order to get protein in denatured condition.
Thank you Adam and Arvinth for your suggestions.
When I did the transfection, I included a GFP control plasmid to see the transfection efficiency because my plasmids are only flag tagged. I aslo did no plasmid transfection by only adding neccessary reagents. The transfection seems to be good and no plasmid control has no bands for either antibody (POI or FLag).
Aravinth thank you again for your suggestions.
First, I recommend that you double-check the sequence to ensure there are no errors that may cause a change in the amino acid sequence.
I believe what Arvinth was suggesting was that if you blot with your antibody against your protein, the FLAG tag should cause a size shift. In that case, you should see two separate bands when you blot with the antibody against your protein. If the tagged version is being expressed, you should see the endogenous protein as one band, and the tagged protein as a slightly higher band.
You need to run the gel for a long while to be able to see small size shifts, but it should be apparent then if two bands are present. If so, then your tag may be misfolded. If not, then the tag is either being cleaved off as Adam suggested, or your entire construct may not be expressed.
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