High Molecular weight reference for Western. Help! - (May/21/2008 )
I am looking at two proteins with a molecular weights of ~130kDa and ~190kDa. All the commonly used reference proteins for equal loading (e.g. Actin, GAPDH) have quite small molecular weights and will run off my gel. Does anyone know of a good higher molecular weight protein (100-250KDa) to use as a loading control (I am working on B cells).
Can you run a gradient gel? That would keep the smaller loading control proteins on the gel with your proteins of interest. Sorry- I know my answer wasn't exactly what you were looking for!
I think gradient gel makes sense.
People often use PonsceauS as "kind of" loading control, not ideal, but worth keeping a scan of PonsceauS staining of the membrane till you find a loading control that suits you best. In the manwhile, strip the membrane and preserve for future western with such loading control.
myosin II heavy chain ~200 kDa
Thanks for all the advice. I think I'll look into doing a gradient gel.
What is PonsceauS? I tried google to no avail!
Try Red Ponceau
It is a red solution that people use to stain their membranes after transfer. I would say it is the equivalent of Coomassie blue, but on the membrane, rather than the gel.
My spellings are generally less clear than a bad western.
My lab uses Hsp90 (90 kD) as a loading control with good success. We use the mouse monoclonal from BD.