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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

CASTING, PREPARING, AND RUNNING SDS-POLYACRYLAMIDE MINI-GELS

Casting, Preparing, and Running SDS-Polyacrylamide Mini-Gels
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
 
Procedure
A. Assembling Gel Molds and Pouring the Resolving Gel

1. Use a clean mold and backing (three blocks). Remove the clamps and the cover from the Hoeffer mini-gel casting apparatus. Do not insert the large block into the mold. Instead, insert only the two small blocks.

2. To pour 0.75 mm gels, lay down a half-piece of wax paper or Parafilm, a glass plate, the 0.75 mm spacers, and another glass plate. Assemble nine more gels in this fashion and stack together. Secure the clamps on the glass plate stack.

3. Prepare the appropriate percentage Polyacrylamide solution for the resolving gel:

To prepare 7.5% SDS-Polyacrylamide gels sufficient for ten 75 mm gels, add the following (see Hint #3):
   25 ml of 30%:0.8% Acrylamide:Bis-Acrylamide (CAUTION! See Hint #1)
   25 mll of 4X Lower Tris Buffer*rxn50 ml of ddH2O
   Final volume is 100 ml
   Add 200 μl of 10% APS

To prepare 10% SDS-Polyacrylamide gels sufficient for ten 75 mm gels, add the following (see Hint #3):
   33.3 ml of 30%:0.8% Acrylamide:Bis-Acrylamide (CAUTION! See Hint #1) 25 ml of 4X Lower Tris Buffer
   41.7 ml of ddH2O
   Final volume is 100 ml
   Add 200 μl of 10% APS

To prepare 12% Polyacrylamide SDS gels sufficient for ten 75 mm gels, add the following (see Hint #3):
   40 ml of 30%:0.8% Acrylamide:Bis-Acrylamide prepared at 4°C
   25 ml of 4X Lower Tris Buffer
   35 ml of ddH2O
   Final volume is 100 ml
   Add 200 μl of 10% APS

4. Add 50 μl of TEMED to the appropriate Polyacrylamide gel solution, mix gently, and immediately pour the gels.

5. Fill a 60 ml (cc) syringe with as much of the Polyacrylamide gel solution as possible.

6. Connect the syringe to a stop-cock, which is attached by a hose to the gel mold.

7. Carefully and slowly dispense the polyacrylamide into the molds. Stop filling each gel when approximatley 3 to 4 cm of space remains above the gel; this is to leave enough space to later pour the stacking gel (see Hint #4).

8. When finished, turn the stop-cock off and take the syringe off. Refill the syringe, if necessary, and continue filling the gel plates.

9. Carefully layer 3 to 4 mm Isobutanol or Tertiary Amyl Alcohol with a Pasteur pipette on all of the gels to prevent oxidation of the polyacrylamide, which inhibits polymerization.

10. Allow the gels to set for approximately 30 min (let polyacrylamide gel waste harden, then throw away).

11. Remove the organic top layer.

12. Rinse the top of the gels approximately five times with ddH2O.

13. Take apart the mold.

14. Cut off the polymerized gel waste at the bottom of the gels with a razor blade.

15. Wrap the gels in Saran Wrap and store at 4°C until needed, or pour the stacking gel.

B. Pouring the Stacking Gel

1. Rinse the top of gel thoroughly with ddH2O.

2. Use a piece of Whatman 3MM paper to dry the inner sides of the plates above the resolving gel thoroughly. Put two clamps securely on the glass plates and stand the gel up.

3. Mix up mini-gel Stacking Solution (1 gel):
   1.8 ml ddH2O
   0.75 ml 4X Upper Tris Buffer
   0.45 ml Acrylamide:Bis-acrylamide (30:0.8)
   20 μl of 10% APS

4. Add 10 μl of TEMED to the mini-gel Stacking Solution, invert to mix, and immediately pour the stacking gel.

5. Rinse a comb (with the appropriate number of teeth for the samples to be run) with 100% Ethanol and wipe clean.

6. Place the comb between the gel plates approximately half-way in.

7. Pour the gel and push the comb in until the top of the teeth is level with the top of the glass plate.

8. Let the gel set for approximately 20 min. Check occasionally to confirm there is no leakage.

C. Setting Up Gel and Preparing Samples

1. Place the gel in a gel-running apparatus (see Hint #5)

2. Pour 1X SDS Running Buffer into the top reservoir and ensure that it does not leak.

3. Pour 1X SDS Running Buffer into the bottom reservoir.

4. To remove bubbles along the bottom edge of the gel, load a syringe (with a bent needle) containing 1X SDS Running Buffer and direct a stream of buffer along the bottom of the gel.

5. Boil protein samples in SDS Sample Buffer for approximately 5 min (do not put samples on ice after boiling).

6. Carefully remove the comb from the gel.

7. Load samples (1 to 10 μl final volume) with a Hamilton microliter syringe. Rinse the syringe four times between samples.

D. Electrophoresis of the Gel

1. Run the gel at 8 to 15 milliamps constant current until the Bromophenol Blue dye front has moved through the stacking gel.

2. Run the gel at 25 to 35 milliamps constant current as the dye front moves through the resolving gel until the dye is just off the bottom of the gel.

3. Take the gel plates apart.

4. Use the gel for Western blot, Coomassie protein stain, or silver protein stain (see Protocol ID#251, Protocol ID#716, or Protocol ID#496).

Solutions
1X SDS Sample Buffer   0.1% (w/v)Bromophenol Blue
Can be stored at room temperature without the DTT. Add DTT just before use.
2% (w/v) SDS
10% (v/v) Glycerol
100 mM DTT
50 mM Tris-Cl, pH 6.8
SDS-Running Buffer (4X)   Store at room temperature
48 g of Tris base
Add ddH2O to 4 liters
4 g SDS
230.4 g Glycine
10% APS   Store at 4°C for several weeks (see Hint #2)
10% (w/v) Ammonium Persulfate
Upper Tris Buffer (4X)   pH to 6.8 using HCl
Add ddH2O to 100 ml
6.06 g of Tris base
Store at 4°C
4 ml of 10% (w/v) SDS
Lower Tris Buffer (4X)   Add ddH2O to 500 ml
Adjust pH to 8.8 using HCl
90.85 g Tris base
Store at 4°C
20 ml of 10% (w/v) SDS
Acrylamide:Bis-Acrylamide   Filter through a Whatman filter #1, then store at 4°C.
Or purchase premixed Acrylamide:Bis-Acrylmide (30:0.8)
0.8% (w/v) Bis-Acrylamide (CAUTION! See Hint #1)
30% (w/v) Acrylamide (CAUTION! See Hint #1)
 
BioReagents and Chemicals
Bromophenol Blue
Hydrochloric Acid
SDS
Glycine
Glycerol
Bis-acrylamide
DTT
Tertiary Amyl Alcohol
Tris-HCl
Isobutanol
TEMED
Ammonium Persulfate
Tris Base
Acrylamide
 
Protocol Hints
1. This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.

2. If the gels are not polymerizing or are polymerizing slower than usual, the problem may be that the APS has gone bad. Make a fresh solution of APS and re-pour the gel.

3. Bisacrylamide forms cross-links in the Acrylamide gel. The APS drives the polymerization of Acrylamide and Bisacrylamide through the production of free radicals. TEMED catalyzes the formation of free radicals from APS and therefore speeds the polymerization process.

4. Don't dispense the Acrylamide too quickly because bubbles will appear and may get into the gels.

5. You may want to use warm Agarose to create a tight seal around the gel. Seal the sides and put a drop of Agarose near each corner.