Transfer high molecular weight proteins - (Jan/15/2006 )

Hi all,

I tried to transfer my interesting protein which has high MW (~270 KDa) to Nitrocellulose membrane. I used the transfer buffer:

Glycine: 39mM
Tris base 148mM
SDS: 0.037%
Methanol: 20%

I transfered with 100mA for 2hours

However I can't receive the signal after transfer.

Is there anyone can tell me the reason and how can i improve it??

Thanks ^^

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did you try all that crazy stuff like checking to see if it was still in your gel, or maybe staining a membrane to be sure it transferred properly?

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I have tried to transfer larger protein too. It is really difficult.
You may try these,
-- Use low % gel, such as 6-8% gel.
-- Transfer at 100V for 2 hours.

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Thanks Patrickn. I'll try again.

I also used 6% of gel and tranfered for 2h; but fixed at 100mA.

@aimikins

I stained the membrane with Ponceau S, but there was no band at the molecular weight I wanted.

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what about staining the gel after transfer? was it still in the gel?

why I am asking, are you sure it didn't blow through?

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