blocking for western blot

[Savita Shah]

Hi:

Try 5% dry milk in TBS (or PBS) containing 0.05% tween 20.. works fantastic for
me.. absolutely no background. If you are working with commercial
his-antibody.. you could use 3% dry milk + 3% BSAin TBS or PBS (containing
0,05% Tween-20).

Hope this helps :)
Good Luck

Savita
spshah at stanford.edu
P:S> I always use 0.05% Tween-20 in every step of immuno-detection protocol..
helps in reducing background

Hope this helps


"Michael L. Sullivan" wrote:

> A collegue once did a sort of study for what blocking agents worked best
> for her situation.  She found (for her Ab) that milk was horrible (very low
> background, but low signal too) and that 3% BSA worked the best (high
> signal, low background).  I suspect that what might be the best blocking
> agent might depend on lots of things, like what the membrane is like, what
> your samples are like, and the nature of your primary and secondary Ab, so
> you might just have to try lots of stuff emperically.  I think some of the
> common blocking agents, are BSA, dry milk, "special" commercial variations
> on dry milk, and tween-20.
>
> Hope this is useful.
>
> Mike
>
> >Dear netters,
> >
> >we are routinely using milk powder as
> >blocking agent for western blots using
> >nitrocellulose blots. However,
> >we have noticed that when we leave out
> >milk powder altogether there is no difference.
> >
> >We now want to try products like super signal
> >from pierce where the company argues that you
> >get stronger and better signals as compared to
> >regular ECL solution. After our first experiments
> >we see that background really becomes a problem
> >with this solution and that we need a good
> >blocking agent.
> >
> >Since we could leave out the milk powder in our
> >protocol without any problems, we start to question
> >whether it has any effect at all. Pierce offers
> >different kinds of blocking agents, but they are
> >expensive and available only in large quantities.
> >
> >We would be glad to hear about any tricks or hints
> >and whether the commercial solutions compare favorably
> >with 'home-made'-recipees.
> >
> >Thanks for your time and help
> >Soenke
>
> ---