A 0.2 M (or 200 mM) stock solution (100x concentrated) of Sodium orthovanadate (Na3VO4) can be prepared by dissolving it in water. This stock solution can be added to the cell lysis buffer to stop protein tyrosine phosphatases.
Reagents and solutions
Sodium orthovanadate (Na3VO4, Mol Wt: 183.91) (e.g., Sigma-Aldrich, #450243)
6M Hydrochloric acid (HCl)
Deionized / Milli-Q water
Equipment and disposables
Falcon® screw cap graduated Conical Tubes, 50 mL (e.g., SARSTEDT, 62.547.254)
Microwave oven/Boiling water bath
Micropipette and tips
0.2 M (or 200 mM) Sodium orthovanadate in water
Preparation of 20 ml of 0.2M Sodium orthovanadate (Na3VO4) solution in water
Use personal protective equipment (lab coat, gloves, goggles, etc) for your safety and follow the guidelines of your institute.
Step 1: Weigh out 0.736 g of sodium orthovanadate (Na3VO4, Mol Wt: 183.91) and transfer it to a 50 mL screw cap graduated polypropylene tube. Add 15 ml Milli-Q water. Tighten the cap and dissolve Sodium orthovanadate by inverting the tube several times. This will take just a few minutes (≈ 5 min).
Sodium orthovanadate dissolves quickly in water. Generally inverting the tube a few times is sufficient to dissolve it.
Step 3: Adjust the pH of the solution to 10.0 using 6M HCl solution.
1. The pH of the solution will be ≈ 13.5. Add 6M HCL solution drop by drop (≈ 10 µl each time) and mix the content by shaking the tube.
2. The solution will turn yellowish as you add HCl.
Step 3: Heat the solution in a boiling water bath for a few minutes. The solution will become colorless upon heating. Stop the heating as soon as the solution turns colorless.
You can also use the microwave to heat the solution. Set the microwave to 800 or 1000 W. Put the tube in a tube stand and place it inside the microwave. Now heat the solution in a cycle of 5 sec. Every 5 sec, open the door and check the color. If the tube lid is closed, open the lid to release the vapor pressure to avoid any blast. You may need to microwave for 15 – 20 seconds to warm the solution.
Step 4: Cool down the solution to room temperature. Readjust the pH to 10.0. Boil the solution as described in step 3. Repeat this (pH adjustment and boiling) another 2-3 times.
You can cool down the tube with running tap water.
Step 5: Adjust the volume to 20 ml with Milli-Q water. Mix by inverting the tube a few times.
Solution can be stored at -20°C in small aliquots (0.5 ml or 1.0 ml).
Throw the solution if you see any color development in the solution.