western blot question

[Pow Joshi]

On 9/25/07, Pow Joshi <pow.joshi from gmail.com> wrote:
>
>
>
> On 9/25/07, Rebecca Pickin <RPickin from cvm.msstate.edu> wrote:
> >
> > Hi everybody,
> >
> > I was wondering if anyone can help me.  I am looking for a breakdown of
> > what bands should appear on a western blot when you load the primary
> > antibody onto the gel.  I understand that to a certain extent various
> > antibodies will be slightly different.  But, since the bulk of the
> > antibody is the same across the board I would expect a certain band
> > pattern.  Does anyone know this or have a reference for this
> > information?
>
>

...I forgot,

you could find the information in Current protocols, or the laboratory
manuals like Antibodies by Ed Harlow and David Lane (this book I find
extensive and lucid).

Hi Rebecca,
>
> all antibodies have a "Y" shaped structure, with heavy chains and light
> chains. If you  use beta-mercapto-ethanol/ DTT etc.,  in your sample, you
> should see the heavy chain bans at around 55kDa. If you have a certain
> isotype (eg:IgG-2b for mouse/ rats etc) you would see a doublet due to
> differential glycosylation of th heavy chains. You should also see a band
> around 25kDa ... these are the light chain bands and they do appear somewhat
> fuzzy or broader. You should also expect a band ~ 150kDa which would be the
> antibody which is not completely reduced.
>
> If you happen to be doing a cross-western after immunoprecipitation, you
> should take care that the protein you are pulling down is not close to these
> relative molecular weights.
>
> Hope that helps
> best,
> Pow
>
> Thanks so much,
> > Becky
> >
> >
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>