Bio-Rad SDS-PAGE Electrophoresis

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Bio-Rad SDS-PAGE

12% Separating Gel Stacking Gel

dI H2O 3.5 ml 6.1 ml

Running Gel Buffer (2) 2.5 ml

Stocking Gel Buffer (3) 2.5 ml

30% T 2.7% (1) 4.0 ml 1.3 ml

10% SDS (4) 100 ul 100 ul

10% Ammonium Persulfate (0.1 g/ml) 50 ul 50 ul

TEMD ___5 ul __10 ul

Total 10 ml 10 ml

Immunoblot

Immunoblot buffer

100 mM Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.5 (adjust pH)

Anti-rabbit IgG (blue) Vector laboratory vectastain ABC kit PK-4001

2 drops in 20 ml immunoblot buffer

AB-kit (mix when membrane soaked with second antibody)

4 drops A reagent

4 drops B reagent

In 20 ml immunoblot buffer

Substrate for ABC-kit

1 ml DAB (40 mg/ml diaminobenzidine tetrahydrochloride)

250 ul of NiCl2 (80 mg/ml NiCl2)

50 ml of 100 mM Tris, pH 7.5

15 ul 30% H2O2

Prepared just prior to staining

Trans-Blot

Transfer buffer

25 mM Tris-base, 192 mM Glycine, 20% v/v Methanol, pH 8.3

(Don’t adjust pH)

3.03 g Tris-base

14.4 g gylcine

Add H2O 700 ml mix

200 ml Methanol

Add dI H2O to 1L

Transfer Power conditions

1.  200 V for 1 hours

2.  a) 100 V 250 mA for 1 hour, or
b) 30 V 40 mA overnight

01/02/07

Bio-Rad SDS-PAGE Electrophoresis

2 of 3

Bio-Rad SDS-PAGE Electrophoresis

Day 1:

  1. Correctly set up the protein gel former. Make sure that it won’t leak later!
  2. Prepare the 12% Separating Gel. Using a Pasteur pipette, add the gel contents to the gel former. Add a few drops of distilled water evenly over the gel.
  3. In about 10-15 minutes, you will notice a transparent line that forms on the gel. This is the sign for you to prepare the Stacking Gel.
  4. Before adding the Stacking Gel, make sure you blot out the water from over the top of the gel.
  5. Once the Stacking Gel has been added, put the combs in.
  6. Once the gel solidifies, set up the gel electrophoresis tank.
  7. Lock the Gel formers into the cassette. Put the cassette into the tank and fill the tank up with TAE?? Buffer.
  8. Carefully remove the combs.
  9. Add 5 ul biotinylated ladder, and 10-15 ul of your prepared samples. Get ice.
  10. Cover the electrophoresis tank. Make sure you connect the red wire to the red side and the black wire to the black side.
  11. Run the gel for ~ 50 minutes at 200 V (don’t change the mA). Make sure to run the reaction over ICE!
  12. While the gel is running, prepare Trans-blot buffer.
  13. Cut filter paper and membrane. For every gel, cut two filter papers slightly bigger than the gel and one membrane as big as the gel itself.
  14. When the gel has finished running, add Trans-blot buffer to a glass Pyrex tray. Soak the filter papers and membrane in this buffer. Make sure the membrane is completely submerged in the buffer!
    NOTE: For Commossie Blue Staining, follow Day 2 for Commossie Blue; For Western Blotting, follow steps 15-25.
  1. Now, set up the transforming tank. Prepare the sandwiches. First, put the sponges on either side, followed by filter papers.
  2. Carefully, remove the gel and submerge it into the Trans-blot buffer.
  3. When ready, put the membrane in between the filter papers on the white side of the sandwich. Make a small slant-cut on the top right corner of the membrane.
  4. Carefully place the gel on top of the membrane, with the marker lane next to the slant-cut edge of the membrane.
  5. Lock the sandwich(es) and place into the transforming tank. Put ice block in the tank.
  6. Run the gel for an hour (60 min) at 100V and 250 mA.
  7. Once the gel has finished running, add enough Tris-Milk into a Tupper-ware container.
  8. After unlocking the sandwich(es), carefully remove the membrane and place it into the Tris-Milk. Incubate with agitation for 30 minutes at room temperature.
  9. When ready, exchange the old Tris-Milk with new one! This time add about 10 ml of Tris-Milk to the same container, making sure that the membrane is completely covered. If not covered, make sure you recalculate the volumes of antibodies to be used.
  10. To the 10 ml Tris-Milk, add 2 ul of antibiotin and 2 ul of appropriate primary antibody (This is 1:5000 dilution).
  11. Leave overnight on shaker in cold room.
  12. Continue with Day 2 for Western Blotting.

Day 2 for Commossie Blue Staining

  1. Remove gel and put into a container containing working stain.
  2. Soak for 1 hour on shaker at RT.
  3. Pour out working stain back into same bottle and pour in Destaining I solution enough so that the gel is covered.
  4. Shake for 15 minutes at RT.
  5. Dump Destaining I solution out and pour in Destaining II solution.
  6. Leave overnight on shaker at RT.
  7. Next Day, cut out a filter paper a little larger than the gel itself, saturate it with distilled water, place the gel carefully on filter paper, cover with saran wrap, and dry the gel at 70˚C for 45 minutes.

Day 2 for Western Blotting

  1. Dump Tris-Milk out. Wash 5 times with 1X TBST (5 minutes each) on shaker.
  2. After last wash, dump 1X TBST out.
  3. Add 10 ml Tris-Milk and to it, add 2 ul antibiotin and 2 ul of secondary antibody.
  4. Leak soak for 1 hour on shaker at RT.
  5. Wash away excess antibodies with 1X TBST (5 minutes).
  6. Make developer (500 ul of each solution on saran wrap, mix well, and place membrane so it is completely saturated with the developer).
  7. Let develop for 5 minutes.
  8. Take a picture! Handle membrane with gloves to avoid fingerprinting.

Taking a picture:

  1. Place membrane on white plateform. Focus and turn white light all the way on! Turn white light button off. Turn Super sensitivity button on.
  2. Acquire.
  3. Movie Setup.
  4. Normal/High.
  5. Exposure Time: 0 Hours, 5-10 minutes.
  6. Total Frames: 6 (Copy to end).
  7. Press GO.

01/02/07