Hi I am doing SDS page analysis in fact western of b-actin from Hepg2 cell lines. I am facing two problems.
1. When I do SDS page the loaded sample moves very slowly. I am using BioRad Miniprotean. I use 30 mA for two gels but the sample doesent move at all !
The value of volt is very low. If I increase the value for the volt to 50 the value of the current increases to 300 mA or more ! I am using Tris-Glycine SDS buffer? It takes a lot of time and it heats up.
2. After transfer primary Ab and secondary Ab a lot of unspecific binding?! In fact all the proteins were stained.