3 replies to this topic

[bsengez]

I have isolated total, nuclear and cytoplasmic proteins from mammalian cell culture stably expressing specific genes. qPCR analysis showed that there is an expression, but I have a western blot problem. I'm using polyclonal primary antibody against my 14 kDa protein. Although my marker run well, my protein samples got stuck at the interface between the stacking and the running gel. I tried to stain my membrane if anything is wrong, but the membrane is totally dark blue. What is the problem? Any help would be appreciated.
Burcu

[mdfenko]

did you block your membrane with protein (bsa, serum, milk, etc) prior to staining?

if you were staining for protein (coomassie, india ink, etc) then the entire membrane will stain if you did.

if immunostaining then the entire membrane will stain if you didn't.

[meaningless]

if you stain your membran with coomassie, i suggest not to stand the membrane for more than 5 to 10 seconds before washing, or else it will be 'over stain' for-ever

[Prep!]

bsengez, on Sep 29 2009, 06:33 PM, said:

I have isolated total, nuclear and cytoplasmic proteins from mammalian cell culture stably expressing specific genes. qPCR analysis showed that there is an expression, but I have a western blot problem. I'm using polyclonal primary antibody against my 14 kDa protein. Although my marker run well, my protein samples got stuck at the interface between the stacking and the running gel. I tried to stain my membrane if anything is wrong, but the membrane is totally dark blue. What is the problem? Any help would be appreciated.
Burcu

if your prootein is stuk between the resolving and stacking, are u sure you are using the right percentage of gel??!!! try staining the gel and confirming if the protein is there. If that is the case may be your conditions need to be more appropriate. Try using methanol in the transfer buffer if you aint!!!