Acknowledgement
The following is adapted from the BIORAD Mini-PROTEAN® 3 Cell Instruction Manual.
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Reagent Preparation and Stock Solutions
Volumes Required Per Gel
The volumes listed are required to completely fill a gel cassette. Amounts may be adjusted depending on the application (with or without comb, with or without stacking gel, etc.).
| Gel Thickness (mm) |
Volume (ml) |
|---|---|
| 0.5 | 2.8 |
| 0.75 | 4.2 |
| 1.0 | 5.6 |
| 1.5 | 8.4 |
Stock Solutions and Buffers
Acrylamide/Bis (30% T, 2.67% C)
87.6 g acrylamide2.4 g N'N'-bis-methylene-acrylamide- Make to 300 ml with deionized water. Filter and store at 4°C in the dark (30 days maximum.)
10% (w/v) SDS
10 g SDS- Dissolve 10 g SDS in 90 ml water with gentle stirring and bring to 100 ml with deionized water.
1.5 M Tris-HCI, pH 8.8
27.23 g Tris base80 ml deionized water- Adjust to pH 8.8 with 6 N HCI. Bring total volume to 150 ml with deionized water and store at 4°C.
0.5 M Tris-HCI, pH 6.8
6 g Tris base60 ml deionized water- Adjust to pH 6.8 with 6 N HCI. Bring total volume to 100 ml with deionized water and store at 4°C.
Sample Buffer (SDS Reducing Buffer)
3.55 ml deionized water1.25 ml 0.5 M Tris-HCI, pH 6.82.5 ml glycerol2.0 ml 10% (w/v) SDS0.2 ml 0.5% (w/v) bromophenol blue-
9.5 ml Total Volume- Store at room temperature. For use: add 50 μl (3-Mercaptoethanol to 950μl sample buffer prior to use. Dilute the sample at least 1:2 with sample buffer and heat at 95°C for 4 minutes.
10 x Electrode (Running) Buffer, pH 8.3 (makes 1 litre)
30.3 g Tris base144.0 g Glycine10.0 g SDS- Dissolve and bring total volume up to 1.000 ml with deionized water. Do not adjust pH with acid or base. Store at 4°C. If precipitation occurs, warm to room temperature before use.
- Use: Dilute 50 ml of l0 x stock with 450 ml deionized water for each electrophoresis run. Mix thoroughly before use.
10% APS (fresh daily)
100 mg ammonium persulfate- Dissolved in 1 ml of deionized water.
Gel Formulations (for 10 ml of gel)
| % Gel | dd.H2O (ml) |
30% Degassed Acrylamide/Bis(ml) |
Gel Buffer(1) (ml) |
10% w/v SDS (ml) |
|---|---|---|---|---|
| 4 | 6.1 | 1.3 | 2.5 | 0.1 |
| 5 | 5.7 | 1.7 | 2.5 | 0.1 |
| 6 | 5.4 | 2.0 | 2.5 | 0.1 |
| 7 | 5.1 | 2.3 | 2.5 | 0.1 |
| 8 | 4.7 | 2.7 | 2.5 | 0.1 |
| 9 | 4.4 | 3.0 | 2.5 | 0.1 |
| 10 | 4.1 | 3.3 | 2.5 | 0.1 |
| 11 | 3.7 | 3.7 | 2.5 | 0.1 |
| 12 | 3.4 | 4.0 | 2.5 | 0.1 |
| 13 | 3.1 | 4.3 | 2.5 | 0.1 |
| 14 | 2.7 | 4.7 | 2.5 | 0.1 |
| 15 | 2.4 | 5.0 | 2.5 | 0.1 |
| 16 | 2.1 | 5.3 | 2.5 | 0.1 |
| 17 | 1.7 | 5.7 | 2.5 | 0.1 |
| (1) Resolving Gel Buffer: 1.5 M TRIS-HCl, pH 8.8 Stacking Gel Buffer: 0.5 M TRIS-HCl, pH 6.8. |
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1. Prepare the monomer solution by mixing all reagents except the TEMED and 10% APS. Degas the mixture for 15 minutes.
2. Immediately prior to pouring the gel, add, for 10 ml monomer solution:
Resolving Gel: 50 μl l0% APS and 5 pl TEMED
Stacking Gel: 50 μl 10% APS and 10 pl TEMED
Swirl gently to initiate polymerization.
Note: Prepare any desired volume of monomer solution by using multiples of the 10 ml recipe. The volumes of APS and TEMED must be adjusted accordingly.
Warning: The catalyst concentration is very important! Webbing and incomplete well formation can result from inaccurate catalyst concentration.
Discontinuous Native PAGE (Ornstein-Davis)
Stock Solutions and Buffers
1. Acrylamide/Bis (30% T, 2.67% C)
87.6 g acrylamide2.4 g N'N'-bis-methylene-acrylamide- Make to 300 ml with deionized water. Filter and store at 4°C in the dark (30 days maximum).
2. 1.5 M Tris-HCI, pH 8.8
27.23g Tris base80 ml deionized water- Adjust to pH 8.8 with 6 N HCI. Bring total volume up to 150 ml with deionized water and store at 4°C.
3. 0.5 M Tris-HCI, pH 6.8
6 g Tris base60 ml deionized water- Adjust to pH 6.8 with 6 N HCI. Bring total volume up to 100 ml with deionized water and store at 4°C.
4. Sample Buffer
5.55 ml deionized water1.25 ml 0.5 M Tris-HCl, pH 6.83.0 ml glycerol0.2 ml 0.5% (w/v) bromphenol blue-
10.0 ml total volume- Store at room temperature.
- Use: Dilute the sample at least 1:2 with sample buffer and heat at 95°C for 4 minutes.
5. 10 x Electrode (Running) Buffer, pH 8.3
30.3 g Tris base144.0 g Glycine- Bring total volume up to 1000 ml with deionized water. Do not adjust pH.
- Usage: Dilute 50 ml of 1.0 x stock with 450 ml deionized water for each electrophoresis run.
Gel Formulations (10 ml)
| % Gel | DDI H2O(ml) | 30% DegassedAcrylamide/Bis(ml) | Gel Buffer(1)(ml) | |
|---|---|---|---|---|
| 4 | 6.2 | 1.3 | 2.5 | |
| 5 | 5.8 | 1.7 | 2.5 | |
| 6 | 5.5 | 2.0 | 2.5 | |
| 7 | 5.2 | 2.3 | 2.5 | |
| 8 | 4.8 | 2.7 | 2.5 | |
| 9 | 4.5 | 3.0 | 2.5 | |
| 10 | 4.2 | 3.3 | 2.5 | |
| (1) Resolving Gel Buffer: 1.5 M Tris-HCl, pH 8.8 Stacking Gel Buffer: 0.5 M Tris-HCl, pH 6.8. |
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1. Prepare the monomer solution by mixing all reagents except the TEMED and 10% APS. Degas the mixture for 15 minutes.
2. Immediately prior to pouring the gel, add, for 10 ml solution:
50 ml APS and
TEMED (5 μl for Resolving Gels; 10 μI TEMED for stacking gels)
Swirl gently to initiate polymerization.
Note: Prepare any desired volume of monomer solution by using multiples of the 10 ml recipe. The volumes of APS and TEMED must be adjusted accordingly.
Continuous Native PAGE
Stock Solutions and Buffers
1. Acrylamide/Bis (30% T, 2.67% C)
87.6 g acrylamide2.4 g N'N'-bis-methylene-acrylamide- Make to 300 ml with deionized water. Filter and store at 4°C in the dark (30 days maximum.)
2. Sample Buffer
1.0 ml Electrophoresis Buffer3.0 ml Glycerol0.2 ml 0.5% Bromophenol Blue5.8 ml Deionized water-
10.0 ml Total Volume
3. Continuous Buffers (McLellan)
McLellan describes various continuous buffer systems from pH 3.8 to pH 10.2. Use the table below to prepare 5 x continuous non-denaturing PAGE electrophoresis buffers. Add both the acidic and basic component to 1 litre of water. Do not adjust the pH. If the final pH is outside the listed range discard the buffer and remake.
| pH | BasicComponent | 5 x Solutiong.l-1 | AcidicComponent | 5 x Solutiong.l-1 |
|---|---|---|---|---|
| 3.8 | β-AlanineMr 89.09 | 13.36 | Lactic acid85% solution | 7.45 |
| 4.4 | β-AlanineMr 89.09 | 35.64 | Acetic acid17.4 M | 11.5 |
| 4.8 | GABAMr 103.1 | 41.24 | Acetic acid17.4 M | 5.75 |
| 6.1 | HistidineMr 155.2 | 23.28 | MESMr 195.2 | 29.5 |
| 6.6 | HistidineMr 155.2 | 19.4 | MOPSMr 209.3 | 31.4 |
| 7.4 | ImidazoleMr 68.08 | 14.64 | HEPESMr 238.33 | 41.7 |
| 8.1 | TrisMr 121.14 | 19.38 | EPPSMr 252.2 | 37.85 |
| 8.7 | TrisMr 121.14 | 30.29 | Boric acidMr 61.83 | 7.73 |
| 9.4 | TrisMr 121.14 | 36.34 | CAPSMr 221.3 | 44.26 |
| 10.2 | Ammonia14.8 M | 12.5 | CAPSMr 221.3 | 22.13 |
| GABA: γ-aminobutyric acid MES: 2-(N-morpholino) ethanesulphonic acid MOPS: 3-(N-morpholino) propanesulphonic acid HEPES: N-2-hydroxyethylpiperazine-N’-2-aminoethanesulphonic acid EPPS: 4-(2-Hydroxyethyl)-1-piperazinesulphonic acid CAPS: N-Cyclohexyl-3-aminopropanesulphonic acid. |
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Dilute 200 ml of 5 x buffer with 800 ml deionized water to prepare 1 x electrophoresis buffer. The final concentrations of buffer components will be:
| pH | Basic Component | AcidicComponent |
|---|---|---|
| 3.8 | 30 mM β-alanine | 20 mM lactic acid |
| 4.4 | 80 mM β-alanine | 40 mM acetic acid |
| 4.8 | 80 mM GABA | 20 mM acetic acid |
| 6.1 | 30 mM histidine | 30 mM MES |
| 6.6 | 25 mM histidine | 30 mM MPOS |
| 7.4 | 43 mM imidazole | 35 mM HEPES |
| 8.1 | 32 mM Tris | 30 mM EPPS |
| 8.7 | 50 mM Tris | 25 mM boric acid |
| 9.4 | 60 mM Tris | 40 mM CAPS |
| 10.2 | 37 mM ammonia | 20 mM CAPS |
Gel Formulations (for 10 ml of gel)
| % Gel | DDI H2O(ml) | 30% Degassed Acrylamide/Bis(ml) | Gel Buffer(1)(ml) |
|---|---|---|---|
| 4 | 6.7 | 1.3 | 2.0 |
| 5 | 6.3 | 1.7 | 2.0 |
| 6 | 6.05 | 2.0 | 2.0 |
1. Prepare the monomer solution by mixing all reagents except the TEMED and 10% APS. Degas the mixture for 15 minutes.
Note: Prepare any desired volume of monomer solution by using multiples of the 10 ml recipe.
2. Immediately prior to pouring the gel, add, for 10 ml monomer solution:
50 μl 10% APS
10 μl TEMED
Swirl gently to initiate polymerization.
Note: Below pH 6, TEMED becomes a less effective catalyst. Increase the concentration of TEMED 5-fold to polymerize gels with a pH range between 4 and 6.
References
- The BIORAD Mini-PROTEAN® 3 Cell Instruction Manual.
- [bibliplug last_name=”Laemmli” year=”1970″]
- [bibliplug last_name=”Ornstein” year=”1964″]
- [bibliplug last_name=”Chrambach” year=”1983″]
- [bibliplug last_name=”McLellan” year=”1982″]
See also:
Electrophoresis and gel staining resources
Electrophoresis and gel staining resources