SDS-PAge problem! - Protein separation (Jun/22/2002 )
Can anyone tell me why I am getting only higher molecular protein bands and none of the middle or low? What could the problem be? HELP.
you have to be clearer about wht you are doing. Are these bands in the gel or on a membrane in a western blot? Are you staining?
may be you have to change your staining solution.
i had exactly the same problem, and I replaced staining solution. and it worked.
maybe you're not running the gel long enough. are you making sure the dye runs all the way at the bottom of the gel.
maybe your ladder is bad...try using a newer one.
Your problem could be that you may not be using the right percentage of PAGE for you separation, for lower MWs you need to use higher percent and also your staining solution may not be right. Hope this helps you
actually the molecular weight on SDS PAGE is not always so accurate, anyway it is not a way to determine the molecular weight of a protein, you need to do gel filtration for accurate weight..in my case I found my protein 10kDa larger but I am sure that is my protein because I have done the functional analysis. Also, have you added the contribution of your vector? The vector may contain large tag.