Im new to western blotting
I have recently tested a new method of isolating cardiomyocytes and extracted protein. Ran a gradient gel (4-15%) of my samples at 10ug protein.
I notice on ponceau staining that it is weak, probably since ive loaded 10ug. I cut the blots as my protein of interest is 80kda and my control gene GAPDH is ~37kDa. I notice on probing for GAPDH that it is slightly larger than 37KDa (its more like 40-45kda). It is extremely hard to probe for my protein of interest as it is weak in these samples
Just wondered whether there was a chance that the protein is degraded some how? If so, would i still be able to use these samples to look at my protein of interest. Or would i just need to load more protein than 10ug
any help is appreciated as im very much a novice at this technique