Note: Make fresh before use Tris pH 6.8 (RT) SDS (RT) Glycerol (RT) 2-ME (4
C) Protease Inhibitor Cocktail 1 (-80
C, aliquots) EDTA (pH 8.0) (RT) NaF (RT) Beta-glycerophosphate (RT) Sodium Orthovanadate 2 (-80
C, aliquots) Sodium Pyrophosphate 3 (4
C) Total Vol
1 Protease Inhibitor Cocktail Stock Conc. 500 mM 20% 500mM 500mM 250mM 200mM 100mM Vol for 1ml of 2x buffer 200 ul 200 ul 100 ul 20 ul 200 ul 20 ul 100 ul 40 ul 20 ul 100 ul
2x conc. 100mM 4% 10% 2% 10mM 50mM 10mM 4mM 10mM 1x conc. 50 mM 2% 5% 1% 5mM 25mM 5mM 2mM 5mM - dissolve one Complete, Mini Tablet (Cat No. 1836153) in 1ml of ddH 2 0 - store cocktail as 100-200 ul aliquots at -80 C - stable for at least 6 months (Roche) 2 Sodium Orthovanadate 3 Sodium Pyrophosphate - stable at -80 C for at least 6 months - needs to be "activated" before use (see below) - if precipitate forms, warm up to RT to dissolve before adding to buffer - stable for at least 4 months
Sodium orthovanadate should be activated for maximal inhibition of protein phosphotyrosyl-phosphatases.
1. Prepare a 200 mM solution of sodium orthovanadate.
2. Adjust the pH to 10.0 using either 1 N NaOH or 1 N HCl. The starting pH of the sodium orthovanadate solution may vary with lots of the chemical. At pH 10.0 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.
6. Store the activated sodium orthovanadate as aliquots at -20¡C.
[Note: This procedure depolymerizes the vanadate, converting it into a more potent inhibitor of protein tyrosine phosphatases.] Reference: Gordon, J., Methods Enzymol. 201: 477-482, 1991