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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Semi-dry transfer of high molecular weight proteins
Posted by: Mats (IP Hidden, New member, 2)
Date: March 24, 2008 08:38AM
Hi,
I am having problems transfering anything above 70 KD on to my PVDF membrane using semidry transfer [1 hour transfer at constant 400 mA]. We are using Taubin's buffer generally. So here is what I have done so far: A) I have added 0.1% SDS to the Taubin recipe [still at 20% methanol] B) I have reduced the methanol from 20% to 10% and even to 0% C) I have increased the transfer time to overnight at 50 mA (~4-10 V). D) I have decided not to equilibrate the membrane in transfer buffer so I keep more of the proteins Nothing works. Time to quit semidry blotting? There has to be SOMEONE out there that knows the sercret to transfering heavy MW proteins with the semidry method. C'mon folks............. Cheers! Edited 2 times. Last edit at 03/24/08 09:27AM by Mats.
Re: Semi-dry transfer of high molecular weight proteins
Posted by: Mats (IP Hidden, New member, 2)
Date: March 24, 2008 08:39AM
8 hours overnight transfer did not help much. Increase to 12-14 hours at the expense of losing smaller proteins?
Edited 1 times. Last edit at 03/24/08 09:26AM by Mats.
Re: Semi-dry transfer of high molecular weight proteins
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: March 24, 2008 05:38PM
There is a old post here: [biowww.net]
Invitrogen has NuPAGE ® Large Protein system that maybe useful. Never tried that. Here is the link: [www.invitrogen.com] Good luck.
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