Help with western blotting - protein not running in to seperating gel (Dec/22/2005 )
Hi I'm looking for help with my blots. I am trying to look for ER alpha in MKN74 cells and the western blots are consistently showing up nice bands but in completlely the wrong place. The proteins seem stuck right at the top of the gel and not running through it. Of intrest I'm running 5% separating gel with 20 mcgs of protein in the wells.
I'd be greatful for any help.
I use 10% gels for ER alpha with 20 mcgs protein. My band comes around 60 kda which is the expected MW. I sonicate my cells in a trition based cell lysis buffer. I have noticed that when I used to use an SDS based cell lysis buffer my cell lysates used to get stuck in the wells.
let me know if u need the recepie..