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SDS-PAGE Gels

We have the mini-protean and criterion system from Biorad.

Criterion system is used for larger gels. Not yet used in Madison, but the basics should be similar to the mini-protean system described below.
See guide for more detail on reagents and protocol.

Mini-Protean system reagents and instructions are listed below.
See guide for more detail on reagents and protocol.

Supplies

Running buffer

Typical running buffer for SDS-PAGE is Tris/Glycine/SDS buffer.

10x Tris/Glycine/SDS buffer
Premade 10x stock from Biorad: 10x Tris/Glycine/SDS #1610732
10x Buffer Recipe:

Amount Reagnet Final Concentration
30g Tris base 25mM
144g Glycine 192mM
10g SDS 0.1% w/v
1L H2O

pH should be 8.3

Dilute 10x stock to make 1x solution. It takes 1250mL (not 1L as stated) to fill the tank up to the top.
- 125mL of 10x stock
- 1125mL of DI H2O

Sample Buffer

All samples should be dissolved/diluted in sample buffer prior to loading

2x Laemmli buffer
Premade from Biorad: 2x Laemmli #1610737. Add 50ul of 2-Mercaptoethanol to 950ul of buffer before use.
2x Buffer Recipe:

Amount Reagnet Final Concentration
10 ml Tris-Cl (1M, pH 6.8) 100 mM
4 g SDS (electrophoresis grade) 4% (w/v)
0.2 g Bromophenol blue 0.2% (w/v)
40 ml Glycerol (50%) 20% (v/v)
** DTT (1M) or B-Me 200 mM

dH2O to 100 ml

** Prepare 2X buffer without reducing agent (DTT or B-Me) and store at room temperature.
DTT: Prepare frozen DTT aliquots (1 M) according to Sambrook & Russell (2001) Appendix I. Immediately before use, prepare 1X reducing sample buffer by mixing 5 ml room temperature stock with 1 ml DTT and 4 ml dH2O. Retain at 4°C and use within 24 hours.

Gel

Gels can be ordered premade from Biorad. Order TGX gels for mini-protean system.
Choose the appropriate gel percentage depending on the size bands you are trying to resolve. Gradient gels should be used if you are trying to separate bands of widely varying molecular weights.

Percent Size
7.5% 40–200 kD
10% 30–150 kD
12% 20–120 kD
4–15% 20–250 kD
4–20% 10–200 kD
Any kD™ 10–100 kD

Ladder

The Mandel lab currently uses the following ladder: Thermo Scientific™ PageRuler™ Prestained 10-180kDa Protein Ladder

Stains

Instead of staining, TGX stain-free gels can be used and bands are visualized fluorescently. NOTE: Denise tried these with our gel doc and it did not work as described. We may be missing the correct filter.

Coomassie Stain

Amount Reagent
0.25 g Coomassie Brilliant Blue (R250)
125 ml Methanol
25 ml Acetic acid
100 ml dH2O

Dissolve stain, then filter with Whatman No. 1 filter paper to remove insoluble material.

Coomassie Destain

Amount Reagent
500 ml Methanol
100 ml Acetic acid
400 ml dH2O

Protocol

Run gel

  1. Unpackage gel - remove green strip from base and remove comb
  2. Set up gel in electrode assembly.
    • If only one gel, use glass casting pieces as buffer dam
    • Make sure wells face inside of the unit
  3. Use green clamps to secure gels
  4. Place electrode assembly into tank
  5. Pour running buffer into tank starting by filling the electrode assembly
    • Electrode assembly should fill up and not leak into tank
    • Wells should be filled with running buffer
    • Once assembly is full, fill tank
  6. Add ladder and samples to wells
  7. Place lid on tank and hook up to HC power supply
  8. Run gel at 200V until dye front reaches the bottom of the gel (30-40 min)
    • Instruction manual says gel can be run at up to 300V, but we do not have HV (high-voltage) power supply for this.
    • If samples do not run evenly, run gel at 50V for about 15min allowing samples to move out of the stacking gel portion of the gel. Increase to 200V once samples have entered the separating gel.
  9. When run is complete, remove gel from apparatus. Use green lever and insert between plastic plates where black arrows indicate.

Stain gel

  1. Stain gel (may need to fix first for tiny bands - see gel guide) for ~30-60 min in Coomassie stain
  2. Image
  3. If gel is needed for further analysis (ie western blot), destain gel. Wash in destaining solution 3x for 15min or until dye is removed.