SDS removal from proteins after PAGE.

[Ed Rybicki]

> Does anybody have any idea - reference or experience - how to remove SDS
> after electrophoresis so that the protein would still be (thoretically)
> active?
>
> Thanks,
> Sagi

Here is part of a protocol from our laboratory manual dealing with
renaturation-in-gel of proteases - hope is useful.

----------------------------------------------------------------------
 .I.GELATIN-CONTAINING POLYACRYLAMIDE GELS FOR THE DETECTION OF
                              PROTEASES;
Author: S. Deane, Dept Microbiology, Univ Cape Town, PB Rondebosch 7700

Pouring, loading and running gels

1) Clamp up Hoefer glass plates using thin spacers.

2) Mix up the gel soln. as follows:

                     8% (Enough for two gels)
      Acrylamide (30% stock)   12    ml
      RGB (=running gel buffer)11.25 ml
      H2O                      17.25 ml
      1% gelatin                4.5  ml
      10% amm. persulfate       0.1  ml

3) cool slightly on ice (not too much or the SDS precipitates)

Washing

     Scrape off the stacking gel, nick the
     top RH corner of the gel (well no.10), then invert it over
     the Triton X-100 (~200 mls) in a tupperware. With the bottom
     edge of the plate in the Triton, wet a spacer with Triton
     and peel back one corner of the gel at the top edge. The gel
     is strong enough to peel back and fall into the Triton
     without breaking! Wash in Triton for 1 h at RT with gentle
     shaking.

Incubating for protease activity

Remove the Triton by suction. Replace with Glycine Incubation
     Buffer (~200 mls), and incubate @ 370 C for 3 h, with gentle
     shaking.

Staining

Remove Inc. buffer and stain ~30` at RT or 370 with Amido Black
     stain, with shaking. Destain in 2-3 changes of destain soln.
     for ~1 h, or O/N. NOTE: gels can be easily re-stained if you
     were too enthusiastic in your destaining, and lost all the
     colour!!

STOCK SOLUTIONS

1% Gelatin
  gelatin      1.0      g
  dH2O        80       ml   } Boil

            Make up to 100 ml with water; autoclave.


Glycine Incubation Buffer 10 X STOCK !!
  Glycine       37.5      g
  dH2O     to  500       ml

            Adjust to pH 9 with 10 N NaOH (~6 ml)
            Autoclave.

Triton X-100 (2.5%)
  Triton X-100 (liquid in weighing room)   25  ml
  dH2O                                 to   1  l

            Stir O/N to dissolve. Autoclaving unnecessary.

Amido Black Stain
  "Amidoschwartz" (for e-phoresis)     1    g
  dH2O                              50   ml
         Mix 10`. Make up to 500 ml with destain. Stir 30`.
         Filter to remove granular matter - very important
         if you don't want blotchy gels.
         (Stain can be re-used several times!)

Destain
  glacial acetic acid       200   ml
  MeOH  (technical)         500   ml
  dH2O                        2   l

          or use the destain in prep. room.
          Destain can also be diluted 1:1 with H2O for
          destaining gelatin gels.

REFERENCES:

see also: Hare,P. Characterization of Extracellular Alkaline
                  Proteases and Collagenase Induction in
                  Vibrio alginolyticus
                  J.Gen.Micro. 129, 1141-1147  (1983)

or the original ref.
          Heussen,C.& Dowdle,E.B. Electrophoretic analysis
                  of plasminogen activators in poly-
                  acrylamide gels containing SDS and co-
                  polymerized substrates.
                  Analyt. Biochem. 102, 196-202  (1980)



  ____________________________________________________________________
 | Ed Rybicki, PhD             |       "Lord, won't you buy me        |
 | (ed at micro.uct.ac.za)        |                                      |
 | Dept Microbiology           |         A Mer-ce-des Benz..."        |
 | University of Cape Town     |                                      |
 | Private Bag, Rondebosch     |                                      |
 | 7700, South Africa          |           - Janis Joplin             |
 | fax: 27-21-650 4023         |                                      |
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