alpha tubulin band the wrong size! - (Jan/29/2008 )
Hi, I've just run a WB on 10 porcine cumulus-oocyte complex just to test if I can detect alpha-tubulins on some recently bought antibody from Sigma (T5168). The Samples were covered in 30ul of Loading buffer + DTT (64mg/ml) and boiled for 5 minutes. The western was done using poured 13.5% separating and 4% stacking gels.
Basically the size that appeared is aproximately 150kDa while the actual MW is supposed to be 50kDa!! So what could have happened? Should I change my sample preparation protocol? Thanks
instead of boiling, try 65C for 10 minutes. you may have aggregated tubulin.
how old is your prep? we found that old tubulin doesn't display a proper banding pattern unless properly denatured in the sample buffer whereas fresh tubulin doesn't even require heating.
how old is your prep? we found that old tubulin doesn't display a proper banding pattern unless properly denatured in the sample buffer whereas fresh tubulin doesn't even require heating.
The cells were frozen for about a week and they were prepared on the day of the western. I shall that method. Thanks!