Disagreement

Phil Whitehead p_whitehead at icrf.icnet.uk
Mon Sep 19 08:58:31 EST 1994


In article <31njhe$fim at highway.LeidenUniv.nl>, sbtnfh at rulsfb.LeidenUniv.nl
(Flip Hoedemaeker, CvF RUL/TNO, Leiden, The Netherlands) wrote:

> In article <199408030058.RAA01171 at net.bio.net>, ERNESTO at VM.CC.PURDUE.EDU (martin) writes:
> >Phil Whitehead wrote that anything below 30 kd will pass through 0.45 nitrocell
> >ulose paper.
> >I donot understand why he is saying that since I have been using nitrocellulose
> > paper with this pore size and found sometimes bands much more lower than my pr
> >otein that is 30 kd (I know that because of non-specific cross reactions with m
> >y antibody). So, basically I do not agree with that point.  Any comments?
> >
> >                                                                    Martin.
> 
> I have not only blotted proteins as small as 6 kd on 0.45 pore size, but I also
> got a good N-terminal sequence from those blots! So I agree with you Martin. 
> 
> For anyone interested, I use Millipore Immobilon P (PVDF membrane), a semi-dry
> blotting device (Novablot, Pharmacia I think), and I don't blot longer than
> 45 mins, because if you blot longer you will lose protein. The same applies for
> nitrocellulose though. Yes, you get hazy bands with such small proteins, but
> they stain with Comassie.
> Flip
>             
>             

While my original statement may have been pedantic I stand by my original
artical with the following qualifications. Proteins are not all alike they
have individual propertiesthe sum of which will dictate their affinity for
nitrocellulose. while individual authors may well have had success with
their own low molecuar weight proteins, IN GENERAL, I still think its a bad
idea to use 0.45 nitrocellulose for proteins below 30K. My own experiance
of calmodulin for instance bears this out- it whistled straight through!
Blotting times methanol concentration will both affect tranfer.
Flip Is using an entirely different matrix in PVDF, this matrix would seem
to give better results than pure nitrocellulose. with PVDF Ican achieve
greater sensitivity particularly when allied to ECL detection.
So to a novice my advice would be If you have a choise use pvdf if not use
nitrocellulose. If youre using nitrocellulose above 30 k use 0.45 pore size
below 30k use 0.22.
 After all you dont want to go through all that gel running and blotting
crap to find that it hasnt worked because your protein has swum off into
the wild blue yonder!


Phil  Whitehead

P.S. Ithink this sort of discussion is just the kind of thing we should be
seeing on the net.  sharing our experiances in this way can only be
helpful.As my old man used to say dont be afraid of making a fool of
yourself by asking questions , its the only way youre gonna learn!



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