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Problem in transfer high MW protein


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5 replies to this topic

#1 sueanne

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Posted 14 April 2010 - 08:12 PM

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

#2 bob1

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Posted 15 April 2010 - 04:52 PM

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

#3 doxorubicin

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Posted 15 April 2010 - 08:09 PM

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

#4 Inmost sun

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Posted 16 April 2010 - 03:07 AM

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

#5 sueanne

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Posted 19 April 2010 - 10:01 PM

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

#6 mdfenko

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Posted 23 April 2010 - 12:07 PM

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