General Support Buffers
| Buffer | Constituents |
|---|---|
| 10X TBE | Tris Base, 216 g; Boric Acid, 110 g; EDTA, 16.6 g QS to 2 L |
| 10X Denaturing Buffer | 0.5 M EDTA, pH 8.0, 20 μL; 1 M Tris-HCl, pH 9.5, 2 mL; 100 mM Spermidine, 1 mL; QS to 10 mL with ddH2O (store at -20 °C) |
| 0.5 M EDTA, pH 8.0 | Disodium ethylenediamine tetraacetate, 186.1 g pH to 8.0 and bring to a final volume of 1 L |
| 10% SDS | Sodium dodecyl sulfate 10 g; QS with ddH2O to 100 mL |
| 20X TAE Buffer | Tris base, 96.9 g; NaOAc-3H2O, 32.8 g; Na2EDTA, 14.9 g Adjust pH to 8.3 with glacial acetic acid QS to 1 L with ddH2O |
| 5X TBE | 0.5M EDTA, pH 8.0, 40 mL; Boric Acid, 27.5 g; Tris Base, 54 g QS to 1 L with dH2O |
| 10X Electrophoresis Buffer | Tris, 30 g; Glycine, 145 g; SDS, 10 g QS to 1 L with H20 |
| Phosphate Buffered Saline | 0.2 M Monobasic Sodium Phosphate, 140 mL; 0.2 M Sodium Phosphate Heptahydrate, 360 mL; NaCl, 8.8 g QS to 1 L with H2O |
| NP-40 Cell Lysis Buffer | Tris-HCl (pH 8.0), 50 nM; NaCl, 150 mM; 1% NP-40 |
| RIPA Buffer | Triton X-100, 1%; Sodium Deoxycholate, 1%; SDS, 0.1%; NaCl, 150 nM; Tris, pH 7.4, 10 mM; PMSF, 1mM |
| 3.2X Sample Buffer | 10% 2-Mercaptoethanol, 1mL; 10% Glycerol, 2 mL; 4% SDS, 4 mL; 0.125 M Tris-Cl pH 6.8, 2.5 mL; H2O, 0.5 mL Aliquot and freeze |
| 10X Running Buffer | Tris base, 30 g; Glycine, 144 g QS to 1 L |
Reducing Sample Buffer
Prepare 2X Sample Buffer (for reducing or non-reducing conditions, dependant on the protein being evaluated) in a 15ml conical centrifuge tube in accordance with the following table:
| Material | Volume Volume (Reducing/non-reducing) |
|---|---|
| Water | NONE/400 µL |
| 0.5M Tris-HCl, pH6.8 | 1 mL/1 mL |
| Glycerol | 800 µL/800 µL |
| 10% (w/v) SDS | 1.6mL/1.6mL |
| 2-mercaptoethanol | 400 µL/ NONE |
| 0.05% (w/v) Bromophenol blue | 400 µL/400 µL |
| TOTAL | 4200 µL (4.2mL) /4200 µL (4.2mL) |
| * Note. A pH of 6.8 is critical for SDS-PAGE. Do not store the buffer longer than 1 month. | |
RIPA Buffer: Preparation of Modified Radioimmunoprecipitation (RIPA)
Buffer
- Tris-HCl (buffering agent prevents protein denaturation)
- NaCl (salt prevents non-specific protein aggregation)
- NP-40 (non-ionic detergent to extract proteins; 10% stock solution in H2O)
- Na-deoxycholate (ionic detergent to extract proteins; 10% stock solution in H2O; protect from light)
Note: Do not add Na-deoxycholate when preparing lysates for kinase assays. Ionic detergents can denature enzymes, causing them to lose activity.
RIPA Protease Inhibitors
- Phenylmethylsulfonyl fluoride (PMSF) (200 mM stock solution in isopropanol; store at room temperature)
- EDTA (calcium chelator; 100 mM stock solution in H2O, pH 7.4)
- Leupeptin (store frozen in aliquots, 1 mg/mL in H2O)
- Aprotinin (store frozen in aliquots, 1 mg/mL in H2O)
- Pepstatin (store frozen in aliquots, 1 mg/mL in methanol)
RIPA Phosphatase Inhibitors
- Activated Na3VO4 (200 mM stock solution in H2O; see Sodium Orthovanadate Activation Protocol)
- NaF (200 mM stock solution: store at room temperature)
Note: Do not add phosphatase inhibitors when preparing lysates for phosphatase assays.
Procedure
Prepare 100 mL modified RIPA buffer as follows:
- Add 790 mg Tris base to 75 mL distilled H2O. Add 900 mg NaCl and stir the solution until all solids are dissolved. Using HCl, adjust the pH to 7.4.
- Add 10 mL of 10% NP-40 to the solution.
- Add 2.5 mL of 10% Na-deoxycholate and stir until solution is clear.
- Add 1 mL of 100 mM EDTA to the solution. Adjust the volume of the solution to 100 mL using a graduated cylinder. Store RIPA buffer at 2–8°C until ready to use.
Ideally, the remaining protease and phosphatase inhibitors should be added to the solution on the same day you are running the assay (100 µL of Aprotinin, Leupeptin, and Pepstatin; 500 µL of PMSF, Na3VO4, and NaF), but with the exception of PMSF the diluted inhibitors are stable in aqueous solution for up to 5 days. PMSF is extremely unstable in aqueous solutions, with a half-life of approximately 30 minutes, and it should be added immediately before use.