Protein blotted membrane storage before probing or .. but how?
John A. Newitt
newitt at nih.gov
Fri Nov 3 19:28:31 EST 1995
In article <47de70$ktv at helka.iif.hu>, jencsa at net.sote.hu (Jeney Csaba) wrote:
> I like to perform a series of experiments which result in a lot of SDS-PAGE
> gels. First I shall use a radioactive method to evaluate them, but for
> confirmation purposes I need to probe them with Mabs according to the Western
> technique later. So the question is how can I store the samples (in gel
> after blotted them to a membrane) without deterioration?
I've done this many times (general protocol below). I can't guarantee
that it will work for every antibody, since it's possible that some
epitopes may be destroyed by the drying process. Of course, if you are
using a McAb the risk is greater if it recogizes such a labile epitope.
With these caveats, I must say it has worked for me every time with the
various antibodies that I've used. This protocol is derived from a paper
by a former colleague (Kaufmann, S.H. et al. "The Erasable Western Blot"
Anal. Biochem. 161:89-95 (1987)).
1. Perform SDS-PAGE and transfer proteins to nitrocellulose.
2. Stain sheet for total protein with Ponceau S or Fast Green FCF, if desired.
3. Rinse sheet times with isotonic buffer (e.g. PBS).
4. Block non-specific protein binding sites with 3-5% nonfat dry milk,
0.1% Triton X-100 (or NP-40) in PBS for about 1 hour.
5. Air dry sheet on paper towels, moving to fresh towel after 5 min to
prevent sticking; when fully dry I keep them in ziplock bags. Keep dry
for as long as you like until you get around to probing with antibodies.
6. Prior to probing with primary antibody, rehydrate the sheet in PBS,
then in blocking solution for a few minutes.
Hope this helps.
John A. Newitt, Ph.D. | <newitt at nih.gov>
National Institutes of Health | FAX: 301-402-0387
Bethesda, Maryland USA |
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