how to do western with 500 kD protein? - (Jun/21/2006 )
Hello everybody, first to say that any help will be highly appriciated since I am quite new in this field! Currently, I am working with transmembrane protein of about 500 kD and my boss wants me to do western blot but in my lab nobody is having any experience with such a big proteins. I discover that 6% SDS is working fine but I am puzzled with transfer. Bands are weak and I am affraid everything stays in gel. Monoclonal antibody as primary is used and appropriate secondary. So, I extended transfer time up to 3h and 100mA. I didn't use Coomassie stain, we don't have it now and it will take a while to order. Any suggestions, anybody with any experience in working with extra big proteins? Thanx in advance....
What I can suggest is to use no more than 10% MetOH in your transfer buffer and no SDS. Then, as I said in another forum, I always stain my gel after transfer if I'm not sure about the transfer efficiency, with Comassie. They also have a solution called PageBlue that is nopt toxic and faster but as sensitive as comassie.
Thanx! This was fast reply! I am using 5% Methanol in my transfer buffer and no SDS at all.
Tris 2.9 g
Glycine 1.46 g
H2O up to 0.5 L
Methanol 25ml
according to the protein blotting handbook, fifth edition (page 15), from millipore, for high molecular weight protein transfers, sds up to 0.05% and prolonged transfer times (up to 21 hours) are recommended.
If today's work doesen't yeld right results I will try with sds and long transfer. What about mA/ voltages with transfer?
Try 20V, transfer in cool room.
I hope this may help.