western blot question

[Pow Joshi]

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?



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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