Problem in transfer high MW protein - (Apr/14/2010 )
Hi there
I'm having problem in transferring the high MW protein onto the blot. I use Towbin buffer with methanol as transfer buffer. I did try prolonged the transfer time to 2h, 150 mA and I still can see some of the proteins in the gel after I stained it. How can I increase the transfer efficiency for the high MW proteins?
Thanks guys
-sueanne-
Long, low, wet transfer is the best. I use 15 v overnight for a tris-glycine system, approx 130 kDa protein.
-bob1-
Some people decrease the methanol from 20% to 10%, and some people add some SDS. 300mA for 2 hours should be the minimum.
-doxorubicin-
sueanne on Apr 15 2010, 04:12 AM said:
Hi there
I'm having problem in transferring the high MW protein onto the blot. I use Towbin buffer with methanol as transfer buffer. I did try prolonged the transfer time to 2h, 150 mA and I still can see some of the proteins in the gel after I stained it. How can I increase the transfer efficiency for the high MW proteins?
Thanks guys
I'm having problem in transferring the high MW protein onto the blot. I use Towbin buffer with methanol as transfer buffer. I did try prolonged the transfer time to 2h, 150 mA and I still can see some of the proteins in the gel after I stained it. How can I increase the transfer efficiency for the high MW proteins?
Thanks guys
as bob1 says overnight blotting is the method of choice; I also recommend to use gradient SDS; the percentage range of AA depends on the molecular mass of interest
-Inmost sun-
Thank you so much guys. Its so useful to me. I will try 
-sueanne-
you can add sds to 0.05%. keep the methanol at 20%.
-mdfenko-