two bands for actin? - (Feb/22/2008 )
Hi!
I'd like to know if it is normal to have two bands for actin. I've tried with 2 antibodies. I had tested in primary cultures of astrocytes and rat vas deferens samples and I got just one band with an antibody (which is not mine) but with my antibody I got two bands for astrocytes and one for rat vas deferens. The two bands are so similar in shape and intensitie and so close in molecular weight that seem to be 2 isoforms of the protein, however I´ve never seen that in papers. It can´t be danified samples because I'had tested the same samples with the two antibodies and the results were the described. And it doesn't seem to be a non specific band, because when I dilute the primary, both bands dissapear. Is there anybody who can help me?
Thank you.
MCQ
Are both of the antibodies recognizing the same isoform of actin? I normally use beta actin as a loading control for western blots.
Well, in both datasheets says that the antibodies recognise a broad range of isoforms, but it doesn´t specify which isoforms. In fact they are both polyclonal antibodies but the blots present in datasheets show just one band for both. I was convinced that this kind of technique didn't allow to distinguish the isoforms (I'm running a 12% polyacrylamide gel) and it would be necessary to run a 2D PAGE (IEF+SDS-Page).
Well, in both datasheets says that the antibodies recognise a broad range of isoforms, but it doesn´t specify which isoforms. In fact they are both polyclonal antibodies but the blots present in datasheets show just one band for both. I was convinced that this kind of technique didn't allow to distinguish the isoforms (I'm running a 12% polyacrylamide gel) and it would be necessary to run a 2D PAGE (IEF+SDS-Page).
Polyclonal antibodies can be notorious for binding to un-related proteins. If there is a monoclonal antibody that you can get your hands on it should help. I have an polyclonal rabbit-anti HIF-1a that binds to Igg's as well as fisher's pre-stained 50kda ladder marker. So no matter what I do I get non-specific bands on places I know there shouldn't be any traces of HIF-1a, and it is more that this polyclonal rabbit antibody have a high affinity to some igg's and BSA. So if i block the membrane with BSA instead of milk, I get a black blot as the primary sticks to everything.