:STAINGEL.SAM

STAINING, DRYING, AND EXPOSING POLYACRYLAMIDE GELS

 

 

N.B.  Use gloves when handling gel.

 

MATERIALS:

 

1.  Coomassie Blue R250

2.  7% Acetic Acid (stock)

3.  (80%) 7% HOAc + 20% methanol

4.  BioRad cellophane membrane backing

5.  Kodak X-OMAT X-AR film

6.  Film cassette with intensifier screen or Phosphoimager cassette

 

PROCEDURE:

 

1.  Disconnect the power supply, remove gel from gel box and separate glass plates to remove gel.  Place gel in a plastic staining box.

2.  Add enough coomassie blue stain to just cover the gel.  Let it sit on rocker for 5 min.

3.  Add enough 7% HOAc to fill the gel box about 1/2 way.  Let the gel rock gently on the rocker.  Continue to destain until gel looks completely destained.  Destaining with (80%) 7% HOAc and 20% methanol can be done after an initial incubation with 7% HOAc. 

     * A quick method of destaining is to place gel in 7% HOAc in the microwave and heat for no more than 10 seconds.*

**For higher percentage gels, soak in 10% glycerol for ~ 1hour before drying**

  4.  Transfer the gel to a metal tray that has been filled with H2O, wet a sheet of cellophane membrane backing and cover both sides of the gel with this membrane.

5.  Use a piece of filter paper to pick up the gel.  Place with the gel on top onto the metal screen of the gel dryer.  Lay the plastic and rubber sheets over the gel. 

6.  Set the dryer and heater times for 2 or more hours.  After creating a vacuum, allow the gel to dry at 80o.  For higher percentage gels, dry at 60o for a longer time.

7.  Once the gel has dried, bring the gel, the film and the cassette to the dark room.  Under the red light (with all doors closed and no other lights on) and with gloved hands, place film on both sides of the gel.  Place the intensifier (shiny side up) on one side of the gel, close the cassette and then wrap the cassette in aluminum foil to prevent light leaks.

      If using a phosphoimager cassette, blank the screen, place gel in cassette, close up cassette and expose for desired time.

8.  To develop the gel, bring cassette to the dark room and under similar conditions as above run the film through film developer.

     If using phosphoimager cassette, remove screen and place in scanner.  Scan the gel into the computer and analyze the data.