Lysis Buffers

Modified RIPA Base Ingredients:

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Tris-HCl (buffering agent prevents protein denaturation)

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NaCl (salt prevents non-specific protein aggregation) br> NP-40 (non-ionic detergent to extract proteins; 10% stock solution in H2O)

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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

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Phenylmethylsulfonyl fluoride (PMSF) (200 mM stock solution in isopropanol; store at room temperature)

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EDTA (calcium chelator; 100 mM stock solution in H2O, pH 7.4)

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Leupeptin (store frozen in aliquots, 1 mg/ml in H2O)

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Aprotinin (store frozen in aliquots, 1 mg/ml in H2O)

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Pepstatin (store frozen in aliquots, 1 mg/ml in methanol)

RIPA Phosphatase Inhibitors

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Activated Na3VO4 (200 mM stock solution in H2O; see Sodium Orthovanadate Activation Protocol)

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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: 1. 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. 2. Add 10 ml of 10% NP-40 to the solution. 3. Add 2.5 ml of 10% Na-deoxycholate and stir until solution is clear. 4. 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. 5. 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. The final concentrations in the modified RIPA buffer should be: · Tris-HCl: 50 mM, pH 7.4 · NP-40: 1% · Na-deoxycholate: 0.25% · NaCl: 150 mM · EDTA: 1 mM · PMSF: 1 mM · Aprotinin, leupeptin, pepstatin: 1 µg/ml each · Na3VO4: 1 mM · NaF: 1 mM

Basic 2X Laemmli Buffer contains: •

4% SDS

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20% glycerol

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10% 2-mercaptoethanol

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0.004% bromphenol blue

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0.125 M Tris HCl

The solution has a pH of approximately 6.8. Other formulations have been devised including: Sample Buffer (4X Laemmli Buffer) •

2.4 ml 1 M Tris pH 6.8 (Same as upper gel buffer)

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0.8 g SDS stock

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4 ml 100% glycerol

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0.01% bromophenol blue. Final Concentration is .02%

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1 ml ß-mercaptoethanol (electrophoresis grade)

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2.8 ml water

IP Lysis Buffer

25mM HEPES, pH 7.4; 150mM NaCl; 1mM EDTA; 0.5% Triton X-100 Stocks to make: 1M HEPES, pH 7.4 0.5M EDTA 0.5M NaCl 500mls Lysis buffer HEPES………12.5ml EDTA………..1ml NaCl………....15ml Triton X-100…2.5ml (1st adjust vol close to final vol, then add last) **Note: Add protease inhibitors before using. Store at 4 degrees.

IP Lysis Wash Buffer

25mM HEPES, pH 7.4; 1M NaCl; 1mM EDTA; 0.5% Triton X-100 500mls Lysis Wash buffer HEPES………12.5ml EDTA………..1ml NaCl………....100ml Triton X-100…2.5ml (1st adjust vol close to final vol, then add last) **Note: Store at 4 degrees.