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wpstep 11-04-2010 07:15 PM

Transfer 2kD peptide
 
I have a 2kD peptide that runs as a Tri-mer at about 7kD....that is what I see with Coomassie staining of gels.I need to optimize the transfer time and voltage but was hoping some one has conditions optimized for a similar size peptide to get me started.
I started with 0.45uM PVDF membrane, 20% methanol in transfer buffer and 25V for two hours.....saw my bands but now they are gone! There was blow through so I am starting with 0.2uM PVDF membrane and a catcher membrane, 20% methanol, 110mA for 2 hours.....will get results later today.
Is there a protocol for working with very small peptide?

wpstep 11-11-2010 05:05 PM

Re: Transfer 2kD peptide
 
Just an update of my progress with this tiny peptide........I am still optimizing the transfer of the peptide but one thing I have learned is that the heat of transfer is not good for something this small....I saw bands when I packed my transfer box on ice!

Mms13 11-14-2012 02:18 PM

Re: Transfer 2kD peptide
 
Hi I am also working with a 1.7 kd protein without much success. I am using a 0.22um nitrocellulose membrane and a 15% tricine gel. I did a 90mA overnight transfer in a 20% methanol buffer at 4 degrees but was not able to see any band. I was wondering if anyone working with small peptides has any tips for me specially on the transfer conditions. Thanks.

DannyB. 01-09-2013 07:36 PM

Re: Transfer 2kD peptide
 
i didnt do any western which such small peptides but maybe this will help you guys maybe the paper "Nishi N. et al. 1995 Western blot analysis of epidermal growth factor using gelatin-coated polyvinylidene difluoride membranes." helps

sry it is not allowed for me to post the link


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