I have constructed two versions of my GOI with and without a FLAG tag and cloned them into the same vector. However, when I transfect HEK293 cells with my two different constructs, I can detect my protein in both cases with an antibody against my protein itself, but I can't get a FLAG tag signal with an anti-FLAG antibody. I even see a little shift of the band of my FLAG tagged protein.
What encourages me is that the FLAG tag is expressed along with the protein. I've previously constructed FLAG tagged proteins in non-mammalian systems and they always worked perfectly fine. The DNA sequences of my constructs are confirmed.
I'm using the anti-FLAG AB from Cell Signaling (1:1000 overnight), which detects the same epitope as Sigma's AB. I do block with 5% milk in PBST. My protein is approx. 15kDa.
Thanks for your help!
Hi Simon,
I had the same problem a few months ago. I had tagged my protein with a C-terminus
Flag but none of my Anti-Flag antibodies recognised the tag. I checked and rechecked the sequence and everything was ok. As Christian mentioned there is a partial renaturation during the transfer of the proteins and due to the fact that the Flag tag is negatively charged it is possible that it could be buried. So bearing this in mind I tried different conditions. It was a headache but I have overcome the problem doing the following:
- Lysis buffer: 20 mM Hepes pH 7.4, 100 mM NaCL, 1,5 mM MgCl2, 1mM EDTA, 1mM EGTA, 0,1% NP40, and your proteases/phosphatases inhibitor cocktails. You will have to pass the extract at least 10 times through a 27G needle.
- Loading buffer: Final: laemly 1X + 20 mM DTT. I did not boil the samples, just leave at room temperature for 5-10 minutes with a brief vortex every 2-3 minutes.
- Transfer buffer: Final: 50 mM Tris pH 8,6-8,8, 384 mM Glycine, 20 % methanol.
These conditions worked in my case, but you can use them as a guide.
Good luck and I hope this was not to late for you.
Jose.
Do you see your protein with the anti-FLAG antibody by other methods, like immuno- fluorescence microscopy for instance? In rare cases the epitope can be masked in the protein as it is on the blot. You completely denature your proteins during SDS-PAGE but they often partially "renature" during transfer or on the membrane. Some domains will fold back to their natural conformation, but most proteins will assume a "random" conformation so it is very well possible that your flag tag is buried and not accessible to the antibody.
I would normally recommend to try an antibody to a different region of the protein but you have already done that successfully. Your FLAG tag represents about 1 epitope so there is not much flexibility.
I would rather play around with electrophoresis and transfer conditions. E.g. go for a native western. I bet that will make your anti FLAG signal appear!
Are you sure that your anti-Flag is still working properly? You should try with another anti Flag. Moreover I think that the Christian's idea to go on IF is nice. I'm using Flag from Sigma. You should do also another test trying the anti flag on a positive ctrl (e.g a flag tagged protein that someone in the lab is using)
If positive controls work and your protein does not work, then maybe the tag was removed by proteolysis. if this is the case you can always change the tag.
Check and recheck your DNA sequence and make sure the FLAG is inframe with your protein. Unfortunately it is also true that some companies sell antibodies that just don't work. Try getting a sample of antibody from another company. As Simone suggested getting a positive control would be good. Another option would be trying to IP with the flag antibody and blot with the protein antibody. Good luck
I also have same problem. I used positive control for FLAG-protein and it works. Although, I can see the protein (~55kDa) in western blot but no FLAG tag (N-terminus) . Actually I tried two vectors pCMV-Tag-2B and pcDNA3 but did not work. So shall I go ahead to try another tag ??
I would really appreciate any help.
Thanks
Hi Simon,
I had the same problem a few months ago. I had tagged my protein with a C-terminus
Flag but none of my Anti-Flag antibodies recognised the tag. I checked and rechecked the sequence and everything was ok. As Christian mentioned there is a partial renaturation during the transfer of the proteins and due to the fact that the Flag tag is negatively charged it is possible that it could be buried. So bearing this in mind I tried different conditions. It was a headache but I have overcome the problem doing the following:
- Lysis buffer: 20 mM Hepes pH 7.4, 100 mM NaCL, 1,5 mM MgCl2, 1mM EDTA, 1mM EGTA, 0,1% NP40, and your proteases/phosphatases inhibitor cocktails. You will have to pass the extract at least 10 times through a 27G needle.
- Loading buffer: Final: laemly 1X + 20 mM DTT. I did not boil the samples, just leave at room temperature for 5-10 minutes with a brief vortex every 2-3 minutes.
- Transfer buffer: Final: 50 mM Tris pH 8,6-8,8, 384 mM Glycine, 20 % methanol.
These conditions worked in my case, but you can use them as a guide.
Good luck and I hope this was not to late for you.
Jose.
Hi Jose, I also have the same problem with flag.
May I ask what is the secret of your protocol?
For the lysis buffer, what makes it different from other routine recipes like RIPA?
For Loading buffer, can DTT be replaced by beta-ME?
Hi Jingjing Jiang,
I recommend you (although you probably have already done it, but just in case):
- Check your cassettes by sequencing.
- Check that your anti-Flag antibody is working using a positive control.
- Check that your transfection protocol is working, cotransfecting with a GFP-containing plasmid for example.
As Christian said (see his answer), during the transfer proteins often partially renature, so (although it is not common, or at least in my experience) the epitope could be buried. The rationale behind my protocol was to extract proteins in a mild lysis buffer to minimize protein unfolding (thus avoiding/minimizing -in part- random folding during the transfer). When I tuned up the conditions I tried RIPA buffer (and others) in parallel, but it does not work in my case, probably because it contains a higher concentration of salts and detergents (including denaturing detergents). I also tried loading samples with and without boiling, and with different concentrations of DTT and without it. Loading protein extracts without boiling does not affect the separation of proteins by western blot (in fact is recommended to detect membrane proteins).
You can use beta-ME instead of DTT, but since beta-mercaptoethanol is a mono-thiol and DTT is a di-thiol, you will need two times more beta-mercaptoethanol molecules to reduce the disulfide linkages between Cys (doubling the unpleasant smell...).
I hope this can help you.
Jose
Hi Jose,
Thank you so much for your detailed reply and explanation.
The sequencing of my plasmids was double checked. I do have the sigma 3Xflag BAP as positive control. I used electroporation in 3T3L1 cell line, which works well.
I have made the buffer following your recipe and I am going to have a try recently. I am going to use beta-ME with double amount as you suggested.
Thanks for sharing your experience!
Jingjing
Hi Jingjing Jiang,
Just bear in mind that to favour protein extraction (since I use a mild lysis buffer), I passed the extract 10 times through a 27G needle.
I also recommend you, if it is possible, to try different conditions (with and without boiling, different amounts of beta-ME). Use my protocol as a guide, because although this work in my case, you could need to tune up for your protein of interest.
Hi Jingjing,
Do you have good news using this protocol? I'm also facing some problems to detect my FLAG-tagged protein in WB.
Cheers,
Rafael
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