Vesicular Monoamine Transporter2 (VMAT2) - (Jun/18/2013 )
I have been doing a lot of Western Blotting for the protein Vesicular Monoamine Transporter2 (VMAT2). I'm currently using a rabbit polyclonal antibody from Millipore. The antibody is very temperamental. Sometimes I get a single band and sometimes multiple bands. Many times I get no signal at all. I keep the experimental conditions same all the time. I even tried making samples without beta-mercaptoethanol and even without boiling. But I can be never sure that I will get any signal. Are there some specific conditions this protein needs to be processed? Or is there a better way to check levels of this protein? Please help
do you get a single band when the vmat2 is fresh, multiple bands as it ages and no bands when it's old?
or do you use freshly prepared vmat2 each time?
have you looked at a stained gel run at the same time as the blotted gel?
have you stained the blot before blocking?
how did they look compared to the immunostained patterns?
Thanks for the reply. Yes, I stain them with Ponceau before blocking. And that looks fine, meaning there is protein on the blot.
Also, regarding the antibody, previously I used to get only 1 band after like 40 minutes of exposure using chemiluminescence. Now I get multiple bands. I have tried using fresh antibody and fresh samples. So its not like the multiple bands are due to degradation products. I don't have problem if there are multiple bands, but the antibody is so temperamental, I can be never sure it will work.
I have tried antibodies from 3 companies, this Millipore one at least works. So I dont know whether trying another antibody will help. Apart from Western blotting, is there some other way I can check levels for VMAT2? Like HPLC? Because we do not have a Mass spec
have you tried elisa?
No I have not. I can try it though. If its not too much to ask for..can you help me with a standard protocol for ELISA? I have never done that technique before. And thanks again for the help
on the top of this page you will see a tab that says "protocol online" (the parent of these fora). click on the tab and you will find a listing of catagories. you can find a few elisa methods under "immunology" (or click this link).
we use elisa to titer our antibodies, not our antigen, so you may not want our procedure.
If I'm understanding correctly and your protein you're trying to detect is in a lysate, you will likely need to do a sandwich ELISA. Typically indirect / direct ELISA's are not sensitive enough to detect a singular protein in a lysate unless it is the predominant protein and your antibody is sensitive.
A sandwich ELISA can be done many different ways and can use the same polyclonal antibody as capture and tracer if some is conjugated to some detection molecule or biotin.
If you would like additional sandwich ELISA information, provide some more information on what your sample are and what you're trying to do and I will try and help you with one of our protocols.
Thank you....and yes my protein is in a lysate. We prepare rat brain striatal synaptosomes by centrifugation and study levels of proteins in it by western blot. I might need what you are saying because my protein is not the predominant one and the antibody does not show any signal at all at low protein concentrations (nothing below 14 ug/lane roughly). What exactly does a sandwich ELISA do?
in a sandwich elisa, you bind an antibody against your protein of interest to the plate, block unused binding sites, apply your mixture so the antibody can fish out your protein of interest, then apply another antibody to your protein of interest and detect.
in other words, you sandwich your protein of interest between 2 antibodies against it.