I made a clone with my insert (230bp) and vector (5kb) which is c-terminally flag tagged. After making the constructs I have checked by sequencing twice! (it`s sure that there is not any stop codon before the flag tag). However, my clones are not working when I run a western blot with them, they are giving correct band when I treat them with the gene-specific antibody but no bands when its treated with the anti-flag antibody (sigma , mouse monoclonal).
I have already tried different batches of antibody with my lab and from some other lab as well.
I have also done immunoflouroscence imaging with flag antibody, though there is high background but still the cells were stained.
I work with whole cell lysate and blot for 2 hours, 300mA, PVDF membrane.
I am not sure what can I do to make it work in western blot. If you experts can help me regarding this, I would be really thankful.
So the antibody is fine for WB.
The fact you have repeated the result in not in contradiction with possible Cter degradation if you have repeated exactly the same experiment (at least it might show you are reliable)... As the tagged protein is detectable in "whole cell" by IM, I would go for a WB performed on a sample made of cells directly solubilized in SDS/PAGE loading buffer in order to avoid extraction. If you get a signal from this, it would tell you there is something occuring during extraction/Lysis (by the way, do you use protease inhibitor cocktail in your lysis buffer?
Maybe your antibody is too diluted. Be careful to use a secondary anti mouse antibody.
Do you have a positive control? Something that has worked before with this antibody?
Hi there,
It might be that this anti-FLAG antibody is OK for IM but not the best for WB or that the FLAG sequence has been degraded during the process of extraction and is not recognized by the antibody anymore...
Thank you all for replying.
Alan.. I have run a positive control as well and it worked fine with the antibody.
Dominique.. that might be a possibility, but I repeated the experiment 3 times and got the same result.
So the antibody is fine for WB.
The fact you have repeated the result in not in contradiction with possible Cter degradation if you have repeated exactly the same experiment (at least it might show you are reliable)... As the tagged protein is detectable in "whole cell" by IM, I would go for a WB performed on a sample made of cells directly solubilized in SDS/PAGE loading buffer in order to avoid extraction. If you get a signal from this, it would tell you there is something occuring during extraction/Lysis (by the way, do you use protease inhibitor cocktail in your lysis buffer?
Dominique - good call about the protease inhibitor cocktail - this could really help if there is some kind of accidental cleavage site around the fusion domain
Thank you again. This I can surely do next time. I added protease inhibitor in the lysis buffer though.
Just a thought: the composition of my lysis buffer is PBS, 1% triton-X and protease inhibitor. Do you think I should change the lysis buffer?
It definitely sounds like your fusion protein is degrading; but it may have nothing to do with protease activity... your tag may be screwing up the fold of the protein and making it prone to mechanical breakdown.
Make sure you do your lysis on ice and try to get your homogenate into SDS sample buffer as quickly as possible.
Is there a reason the tag has to be C-terminal? It might work better for this protein if it is N-terminal.
You said the insert is in right reading frame, the expression is detectable in immunoblotting by protein specific antibody, anti-FLAG antibody works with positive control. Then it make sure your protease inhibitor mix is working OK, it is possible that the tag might have cleaved from the protein.
Also make sure of using the cell line that doesn't express the protein you are interested in for transfection, which will confirm whether the expression you are detecting with the protein specific antibody is not due to endogenous expression of the protein.
Are you able to make this flag-tag Ab work on Western blots with other tagged targets?
I am wondering if maybe it's the blocking buffer you're using for Westerns. You'd be amazed how much blocker choice affect Ab specificity. I've seen primary antibodies that clearly "don't work" (no band) actually work just fine with a different blocking agent. It's worth a try.
Amy.. Thanks for replying. as a matter of fact, I am still struggling with it, I tried 5% and 3% non-fat milk with PBST as blocking .. tried native PAGE also.. but still no result. I am now thinking of changing the vector from1x flag to 3x flag.
But what blocking do you suggest g in this case?
If I remember correctly, you said positive control worked fine in your Westerns. So I don't think blocking is the problem in your case.
I have the same issue, and have seen people on other sites have the same issue. In our case we've made a protein with a 3xFLAG in-frame in the middle of the protein, and another tag at the C-term. The protein has the expected MW shift from the FLAG tag compared to the same fusion without the FLAG and is recognized by western with the other tag. I know our FLAG antibody works with many other fusions in western blot under many conditions, but it doesn't with this particular fusion. I'm wondering if the FLAG epitope is inaccessible in certain fusions depending on how the protein binds to the membrane. The one common thread I can see in all the people having this issue is that they're using PVDF, including us. Since nitrocellulose and PVDF bind the proteins in different ways, maybe the FLAG sometimes isn't accessible on PVDF? I'm going to try nitro and see how it goes.
This reply is long after your question, and I hope you found your solution by now!
To check in, we've verified that the FLAG antibody isn't recognizing certain FLAG tag-containing bands on the blot. In single lanes, some bands are detected while others aren't. We've used an antibody to the 2nd tag in the polypeptide (detects all bands) as well as a polyclonal to the entire fragment (detects all bands) . The FLAG epitope must be inaccessible due to amino acid context and membrane binding on the PVDF.
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