Protein staining with KCl and electroelution

marder at agri.huji.ac.il marder at agri.huji.ac.il
Thu May 6 03:40:22 EST 1993


In article <9305051350.AA27328 at server.uga.edu>, mcgraw%gandal.dnet at SERVER.UGA.EDU writes:
>
> Hi Netfolks,
>
> Anybody out there have experience staining protein gels with KCl and then
> electroeluting the band you want?
>
> This method was mentioned in a paper, but no details or reference were
> given.  Maybe this is common knowledge for some people, but obviously
> not for me.  They did say they used 25 mM KCl to "stain."  I imagine
> you'd get white bands, but...What's the sensitivity?  Any tricks to it?
The white bands are due to localised precipitation of SDS.  The technique
is not very sensitive, and to see the band, I find that the best way is to
put the gel on a BLACK background and illuminated from an oblique angle.
BTW, the band will only be visible for a short time!
Actually, my own experience is with the reverse procedure using 4M Sodium
acetate to stain.  The protein bands are clear against a white background.

>
> Also, assuming I can see something and cut it out, what's the best way
> to electroelute the protein?
One way is to equilibrate in electrophoresis sample buffer (you can drop
the SDS conc. and omit bromophenol and glycerol) and then elute in
Tris-Glycine running buffer.  Put the gel strip in a dialysis bag, lay it
across a flat-bed electrophoresis setup and give it 30-60 minutes at up to
10 Volts per cm (between electrodes).  When you are done, reverse the field
for about 30 seconds (to unstick protein from the dialysis bag) and empty
the contents of the bag into a test tube.  You may want to precipitate the
protein with 10 per cent TCA.

>
> How much protein can one hope to recover from a gel slice?  Assuming it's
> a fairly big (20cm x 20 cm x 1 mm) gel, how much should one load?
You could probably get up to 1 mg.

>
> I want to use the protein for making antibodies.  I'm running the gel
> right now.  Any comments, especially about the staining, would be
> greatly appreciated.
>
> Thanks,
>
> Al McGraw
> mcgraw at bscf.uga.edu
--
                                 '      Jonathan B. Marder
Internet: MARDER at AGRI.HUJI.AC.IL |      Department of Agricultural Botany
Bitnet:   MARDER at HUJIAGRI        | /\/  The Hebrew University of Jerusalem
Phone:    (08 or +9728) 481918   |/  \  Faculty of Agriculture
Fax:      (08 or +9728) 467763   /      P.O.Box 12, Rehovot 76100, ISRAEL



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