Hello Md Abu Siddique

For a 6-well plate, you will require 150-200 µl of lysis buffer per well. Always prepare ~10% extra volume of lysis buffer. Add protease and phosphatase inhibitors fresh to the lysis buffer just before carrying out cell lysis.

You may prepare stock solutions of protease and phosphatase inhibitors.

Protease Inhibitors:

Aprotinin: Dissolve 20 mg in 10 ml of water or PBS to get 2mg/ml stock (1000X). Working concentration is 2µg/ml, so add 1µl of stock in 1 ml of lysis buffer.

Leupeptin: Dissolve 50 mg in 10.5 ml of water to get 10 mM stock (1000X). Working concentration is 10 µM, so add 1µl of stock in 1 ml of lysis buffer.

Pepstatin: Dissolve 5 mg in 7.3 ml of Methanol or DMSO to get 1 mM stock (1000X). Working concentration is 1µM, so add 1µl of stock in 1 ml of lysis buffer.

PMSF: Add 17.4 mg of PMSF per ml of isopropanol to prepare 100 mM solution. Store at −20°C. PMSF is inactivated in aqueous solutions. Use 0.1-1mM in final volume of your lysis buffer.

Phosphatase inhibitors

Sodium orthovanadate

1) Prepare a 100 mM solution of sodium orthovanadate. For preparing a 50-ml stock solution of 100mM sodium orthovanadate, add 0.92 g of powder to 30 ml water.

2) Adjust the pH to 10.0 using either 1N NaOH, or 1N HCl. The starting pH of the solution may vary depending on the lot of the chemical. At pH10.0, the solution will be yellow.

3) Boil the solution until it turns colorless (approximately 10 minutes).

4) Cool to room temperature.

5) Readjust the pH to 10.0 and repeat steps 3 and 4 until the solution remains colorless and the pH stabilizes at 10.0. Make up the volume to 50 ml with water.

6) Aliquot and store the activated sodium orthovanadate at -20 degree C.

Working concentration is 2mM, so add 20µl of stock in 1 ml lysis buffer.

Sodium Fluoride: Dissolve 41.98mg in 1ml distilled water to get 1000mM stock. Working concentration is 10mM, so add 10 µl of stock in 1 ml lysis buffer.

You may also refer to the link below for other commonly used protease and phosphatase inhibitors.

https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/protease-phosphatase-inhibitors.html#:~:text=The%20resulting%20unregulated%20proteolytic%20activity,and%20activity%20following%20cell%20lysis.

You may also use protease/phosphatase inhibitor cocktail available commercially provided as a 100X stock solution. Dilute the product into desired lysis buffer to obtain a 1X concentration.

Best.