Big protein - (Nov/27/2006 )

I want to detect the activation (phosphorylated form) of a big protein (460kDa) by western blot.
I have been working with smaller proteins (about 60kDa) and in that case I have had good results loading 50 ug protein in 10% pre-cast gels that I transfered at 70V for 1 hr.
In this case I am using a 5% acrylamide pre-cast gel, added SDS to the transfer buffer and transfered (in a tank system, nitrocellulose membrane) one hour at 100V, but my Ponceau S staining is very bad and even I got bands I can not be sure those difference are real or just a transfer problem.
I would appreciate any suggestions.

hi,

For larger proteins I always transfer overnight at 35 V (using Biorad system)
followed by 100V for 1 hr the next morning. This works exceptionally well.
I have had alot of problems trying to detect large proteins after a one hour
transfer.

Thank you Minnie Mouse and Minkew. I will tray the over night transfer (crossing my fingers).
I have another question. Since this is a big protein, the running conditions should also be modified? I usually run the gels at 100V until the loading dye reaches de bottom. Should I run the gel a lower voltage to improve the separation and get all the protein leave the wall?.
Thank you.

Can you give me the complete protocol and buffers that you use to transfer your large proteins, please?
Thanks a lot

a 24h RT semidrytransfert gave e the best results for a 250kD protein on nitrocellulose membrane. Didn't tried with PVDF membrane.
I think you should follow your migration by prestained marker and let more migration than just the blue reaches the end of gel. Generally, it means that for a 8cmresolving minigel, the 250kD protein is just 4-5mm below the stacking. So i gues maybe a 400kD just entered the gel.
Moreover, transferts are generally of little lower efficiency at borders of the gel.