Protein free blocking agent
Dr.Narayana R.Isola
(nitrocellulose, Nylon) during pre hybridization?? I would apprecieate
recieving information regarding this.
Thanks,
Narayana R.Isola Ph.D.,
Oak Ridge National Laboratory,
Oak Ridge, Tennessee 37831-6378
Tel 423 74 5893
Hiranya S. Roychowdhury
Well, if you are talking about nucleic acids on NC or Nylon, salmon sperm
DNA and yeast t-RNA are routinely used as blocking agents (for all kinds of
hyb. buffers) and these are non-proteinaceous. Church & Gilbert's phosphate
buffer, with 7% SDS, has been used without the conventional use of any
blocking agents.
For western blots (NC, PVDF, etc.), usually BSA or dried milk powder is
used. I wonder if SS-DNA can substitute for these.
Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroy...@nmsu.edu
Ina Hinners
protein-free blocking agents for Western. I never tried them though, and
I dont know if they'd let you know what it is made of....
Hope this helps, Ina
--
Ina Hinners
ICRF
Secretory Pathways Laboratory
44 Lincolns Inn Fields
WC2A 3PX London
UK
email: I.Hi...@icrf.icnet.uk
Deacon Sweeney
Deacon Sweeney
Fred
blots. Works extremely well. Never tried it for hybridisations, but
should work equally well (I would guess).
see: Haycock, J.W. (1993) Polyvinylpyrrolidone as a blocking agent in
immunochemical studies. Anal.Biochem. 208, 397-399.
Phil
Nick Theodorakis
wrote:
> We use PVP-40 (very cheap from Sigma) to block NC or PVDF for
> western
> blots. Works extremely well. Never tried it for hybridisations,
> but
> should work equally well (I would guess).
[snip]
Well, it is one of the ingredients of Denhardt's solution, after all.
Nick
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Stephen Dahl
Fred <dyn...@hotmail.com> wrote in message
news:38195DF5...@hotmail.com...
> We use PVP-40 (very cheap from Sigma) to block NC or PVDF for western
> blots. Works extremely well. Never tried it for hybridisations, but
> should work equally well (I would guess).
>
> immunochemical studies. Anal.Biochem. 208, 397-399.
Interesting. I saw this paper, tried it, and found it to work extremely not
so well. Did you A/B it with gelatin, BSA, milk, etc.? I'd be interested
in whether you altered the protocol at all. In my hands, coomassie blue
would have given the same results. Are you doing chemi or colorimetric?
Regards,
Steve Dahl
JHMI
ste...@jhmi.edu
Fred
noted a problem, having used it for about 6 yrs. Still using it now.
No other changes to standard blot protocols. An advantage of the PVP40
is you can amido black (or coomassie) stain after chemi, rather than
before. We find this very useful (still works after milk blocking, but
not so well).
whats' "A/B"? We switched to PVP40 from skim milk.
Phil
Stephen Dahl
> Funny. We use NC and PVDF both. Exclusively use chemi. Never really
> noted a problem, having used it for about 6 yrs. Still using it now.
> No other changes to standard blot protocols. An advantage of the PVP40
> is you can amido black (or coomassie) stain after chemi, rather than
> before. We find this very useful (still works after milk blocking, but
> not so well).
> whats' "A/B"? We switched to PVP40 from skim milk.
Sorry, a little jargon there. A/B is simply comparing something side by
side. When I did my test I ran a mini "curtain" gel (More jargon,! Put the
comb in teeth up--not too deep--and after polymerization you have one well
that extends the width of the gel) I ran a cell lysate, blotted and then
sliced strips with a razor blade. This yields identical blots for
comparison. Then you can either use the plastic bubble package from Qiagen
mini columns or the cases from coverslip boxes or anything you've got that
works with small strips individually to compare blocking agents. I checked
out milk, gelatin, BSA, PVP40 and got best results with milk. You've
piqued my curiosity though so maybe I'll try it again with a different
primary antibody.