Hi all,
Whenever I try to do immunoblot for FLAG or HA tag, I could not able to see their expression. Do I need to modify my protocol? I am successfully getting images for endogenous expression.
Please check my brief protocol
I have purchased at least 10 different overexpression construct of my genes that has Flag-HA tag. I transfected the construct(s) in HEK293T cells and harvested after 48 h. I lysed my samples with RIPA buffer (protease inhibitor added) and loaded in the relevant SDS-PAGE. I performed wet transfer method to transfer my protein of interest to PVDF membrane. I have blocked with 5% skim milk for 1 h followed by overnight primary antibody (anti-flag 1:1000) at 4 deg C. Next day, three times wash with TBST and secondary ab (1:10000) for 1 h RT and wash 3 times. Pictures have been developed using ECL substrate and imaged by ChemiDoc.
TIA
Arun.
Based on what you've described:
your extracts, SDS-PAGE, antibody, WB etc..are good because you can detect the endogenous form of your protein. Thus, the problem should occur at another level i.e. upstream those steps.
Maybe, if you cannot detect your proteins it is because they are not expressed!
What about your transfection? have you got a control.
I would suggest you to co-transfect with your constructs a GFP or any other fluorescently tagged protein and inspect visually with a microscope your transfection efficiency.
You could also double check your constructs: mammalian promoter, insert in frame etc...
I have just overlooked something:
what kind of Antibodies are you using to detect the tagged prot?
anti-HA or Flag or an Ab specific for the protein? Anti-HA or even Flag are sometimes crappy. Make sure those Ab are functional.
Thanks for your feedback.
My control transfection (RFP) is working and efficiency is around 80-90% in HEK293T cells.
The promoter is CMV and I have already sequenced full length. There is no problem with the frame shift mutation.
I have tried both anti-HA and anti-FLAG. Both of them are troubling me. But the same antibodies (HA and FLAG) are working for some of my other overexpression plasmids.
You mentioned the endogenous level of your POI. Check your transfected samples by that ab. Actually, you should see some intense extra signal corresponding the construct (assuming the ab works on it). WB is the best for this.
I have tried that also. I could not observe additional band for transfected samples using endogenous ab.
Than there is no active transcription. Call back the Company and complain. Sorry. Good luck!
Note the primary antibody from Mouse or Rabbit? And the source of secondary antibody should correspond to the mouse or Rabbit. Good luck!
We express HA- and Myc-tagged proteins on a regular basis, detection in our hand is never the problem, however, no expression is the common issue. Just because you sequenced your construct and there is no mutation, it does not guarantee transcription of the construct and/or translation of your transgene mRNA. Assuming that your recombinant protein with the tag can be detected with the antibody specific for the protein of interest, you already mentioned you cannot see any additional bands or higher levels of the endogenous protein, which is a give away that you need to look at the steps before protein synthesis. I suggest hat you look for the mRNA by PCR using one of the primer for the tag (HA or FLAG) and the other for the gene. Also, I suggest that you use immunostaining to detect the recombinant protein (anti-HA or anti-FLAG) in transfected HEK cells, and with these two assays you should be able to pinpoint the problem. Good luck.
I suggest to check your construct by restriction and or sequencing
Although it sounds like no expression could be a possibility, depending on the protein you are studying, you could have expression yet due to cellular regulation of protein levels you are not seeing it. Are you trying to observe a protein whose concentrations are tightly regulated? If so, you may not notice an increase in concentration when detecting the HA- or FLAG-tagged version using Ab that detects the endogenous Ab. So, you might want to try increasing the HA/FLAG Ab concentration to 1:500 or even 1:200 just to get a signal (or verify a signal is unobtainable). Alternatively, is your FLAG- or HA-tag possibly being removed/cleaved? Is it preceding a signal sequence and therefore being eliminated from the mature protein? Or, is your protein known to be post-translationally modified by proteases? Have you tested tagged versions of your protein where the FLAG or HA epitope is on either the N- or the C-terminus?
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