SDS-PAGE of protein from membrane fraction - problems with protein band profile (Mar/15/2005 )
I am preparing membrane proteins, briefly, as follow: cells monolayers are washed with PBS and then resuspended in 10 mM TrisHCl, pH 7.4 and 250 mM sucrose plus protease inhibitors. Then, membrane fraction is collected after ultracentrifugation from the interface of a 50 % sucrose cushion.
1- My goal is to run SDS-gels and cut the high molecular weight bands (around 180 kD) for LC-MS analysis.
2- The problem: After staining with colloidal coomassie the samples (prepared simply by boiling 5 min in Laemmeli sample buffer) run as a blue smeary background and on top some bands appear rather faint. I would expect to see more bands but I believe because of the blue background I cannot distinguish the less intense protein bands.
3- Questions: Why the blue background all over the lane? Why so few visible bands? Could other interfering membrane components present in the sample cause the smear?
Please, I greatly appreciate your suggestions and imputs.
load less sample, destain for longer time.
also, you can preclean your sample before loading, maybe aceton precipitation would rid your sample of contaminants.
if you are interested of the top band, run your gel for longer time at low voltage.