5 replies to this topic

[sueanne]

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

[bob1]

Long, low, wet transfer is the best.  I use 15 v overnight for a tris-glycine system, approx 130 kDa protein.

[doxorubicin]

Some people decrease the methanol from 20% to 10%, and some people add some SDS.  300mA for 2 hours should be the minimum.

[Inmost sun]

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

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

[sueanne]

Thank you so much guys. Its so useful to me. I will try  

[mdfenko]

you can add sds to 0.05%. keep the methanol at 20%.