Silver staining Western membranes

I routinely stain Immobilon P membranes used for Western bloting with
Coomassie R250 after finishing immunodetection (in case I don't need to
reprobe them in the future. I wonder if someone ever successfully tried
silver staining such membranes instead? Also, can membranes be reprobed
after such staining (I believe they cannot be after Coomassie staining)? I
thought there was a discussion about this here in the past, but I cannot
find the thread in the archives.
Emir

>I routinely stain Immobilon P membranes used for Western bloting with

I don't see why not, although I've never tried it. I've seen a
protocol in Analytical Biochemistry for transferring proteins for
Western after Coommassie staining the gels, so there is nothing
inherent in the staining that would prohibit it. I suppose it depends
on the sensitivity of your antigen.

Sean
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"Sean Patterson" <[Only registered users see links. ].edu.ar> wrote in message
news:a05010405bb98c98b8dba@[10.0.10.84]...
I

Well, I just tried it, and it did not work. Now I think it is because the
process of siver staining is not exactly similar to Coomassie. The dye binds
to protein, whereas Ag precipitates around protein and thus gets washed
away, which would not happen if protein was immobilized in a gel rather than
on a membrane. Anyway, I have been able to Coomassie-stain my membranes
after unsuccessful Ag-staining attempt.
....Emir

If staining the western blot is being done for record keeping or for
analytical purposes, have you considered ponceau-S staining. It is definitely
not as sensitive as silver staining, but the sensitivity is nothing to scoff
at either. It is comparable to CBB-R. And, it is completely reversible
leaving a blot ready for further downstream processes. It works fine on NC
and PVDF. Have not tried on immobilon-p, etc.


Quoting EK <[Only registered users see links. ]>:



--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---

It also works on Immobilon membranes.

Dr. Hiranya S. Roychowdhury wrote:

>"Sean Patterson" <[Only registered users see links. ].edu.ar> wrote in message

Sorry, I didn't express myself clearly. I was referring to Westerns
after Coommassie staining, not silver.

Sean
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"Sean Patterson" <[Only registered users see links. ].edu.ar> wrote in message
news:a05010400bb99e81ac93e@[10.0.10.84]...
to
tried
reprobed
staining)?
cannot
binds
than

No problem. :-)

By the way, Coomassie stain takes some knowledge to do. Here is my protocol.
Take the wet membrane (after Western it is probably still in TBST.1), rinse
briefly with water. Stain membrane in Coomassie fast stain solution
(0.1%Coomassie R-250 in 10% acetic acid, 40% ethanol) until it turns dark
blue (at least 15 min). Pour off the staining solution and destain briefly
with fast destain (same as above, but without Coomassie). Remove membrane
from destaining solution as soon as you start seing protein bands. Put the
membrane on the filter paper and let dry. I was able to detect down to
0.1-0.2 ng (I assume all antibodies get stripped away by low pH of acetate).

By the way, compared to Coomassie stain above, Ponseau red sucks :-),
otherwise I would use it.
- Emir

Never did a western transfer after staining with CBB-R. I have not done it
because I believe the blot will be useless for proper immunodetection if the
polypeptides are stained. However, Transferring ponceau stained gels has
worked.



Quoting Sean Patterson <[Only registered users see links. ].edu.ar>:



--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---

In article <X0Ycb.42$%[Only registered users see links. ]>, "EK"
<[Only registered users see links. ]> wrote:
).


Try Aurodye. It is quite sensitive. It used to be made by Amersham. I
have not kept track of who owns them now, but a search for Aurodye should
do the trick. It is quite expensive.


Roland

>Never did a western transfer after staining with CBB-R. I have not done it

If I recall correctly, the gels were completely destained before
transfer, but it's been so long since I looked at the original paper
that I'm really not sure.

Sean
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