antiFLAG detect non specific band not FLAG - (Feb/13/2006 )
dear all,
Anybody use antiFLAG antibody? I use antiFLAG antibody from sigma, mouse antiFLAG. then detect my fusion protein FLAG-my protein. supposed about 68kDa, but now it detected a band about 80kDa in all sample include a control without my protein only FLAG. very strange. can anyone give me a hint? by the way, I use antimy protein detect the right size band both control and fusion protein
Thanks a lot.
Hi-
yeah the antiFLAG Antibodies from Sigma suck big time. We recently got some of the newer, affinity purified monoclonal, which in my hand only binds 3 rather than 6 non specific bands by western at a fairly dilute concentration (1:4,000 from 1mg/ml). If anyone knows a way to trouleshoot this I would like to know as well as I have the same problem. I can detect my protein off lysates but not with antiFLAG despite the epitope tag, which I'm sure is in frame.
Am I wrong or is it abnormal to have to affinity purify a monoclonal antibody? I think that may say something about the quality of the antiFLAG ab's from Sigma.
really sucks this antibody. I read couple of papers and they do use sigma antiFLAG antibody and seems good band. why here not work. very bad. any one has idea. appreciate about it.
Time for the forum moderator (me) to weigh in on this..particularly because I have a paper out using this antibody, and had quite a few problems initially.
First off, the FLAG mAb I used was from Stratagene..the M2 clone (not calcium sensitive, the M5 clone I believe is the calcium sensitive clone). The M2's epitope is not entirely unique. Five of the amino acids in the FLAG M2 epitope region (I believe the first 5 aa) belong unfortunately to the Mg-dependent beta phosphatase family of enzymes. To make matters worse, the sequence is upstream of a splice site, and this family is supposed to many splice variants.
For proof of cross-reactivity, I would contact Sigma, and ask for a guy with a last name of Brizzard I believe. I have a copy of his paper (packed away) where they clearly report on cross reactivity of this "unique" epitope.
I wouldn't use it if possible. However the plus side of this epitope is that it's very small. I have been successful in using it..But I will say that some people have published some papers using the M2 clone and were far more successful than even myself
Your success in it working depends on your samples, the application, and the prevalence of your protein. If you have a weakly expressed protein for IHC it may not work too well, however if you have a reasonably good amount, it may just work. For IHC at the minimum I would suggest purchasing the immunizing peptide so you have no doubt in your observed signal.
Message me if you have questions...
thanks viper. seems lot info you give. I did not get it that you suggest purchasin the imunizing peptide, the sigma one M2 antibody isn,t imunizing one?
I am confused.
sorry, maybe a stupid question. hehe
I am confused.
sorry, maybe a stupid question. hehe
Oh one more thing, the cross reactivity of was determined by dot blot. No molecular masses were given. But you could look up the masses of the protein family I mentioned above
I would bet maybe some members of that enzyme family have masses at ~42 and 35 kDa, as these are the background bands I pick up on western with anti-FLAG
Do they detect any 19 and 15 KDa protein using anti-flag M2 Sigma ???
The Flag antibody is not specific especially in IHC. I had huge problem when we tried to it on rat brain. The anibosy might work depending on the sample being tested.
I am trying to detect my flag-tagged protein by western-blot. However, my negative control is positive. It means that I have a nonspecific band at the same size of my interesting protein. So I am trying to dicover if somebody have notice a nonspecific band with aproximatelly 15-20KDa or it happens only with me.
I will appreciate any comment.
