Note: Make fresh before use
3 Sodium Pyrophosphate
Stock Conc. Vol for 1ml of 2x buffer
- if precipitate forms, warm up to RT to dissolve before adding to buffer
- stable for at least 4 months
2x conc. 1x conc.
Tris pH 6.8 (RT)
SDS (RT)
Glycerol (RT)
2-ME (4

C)
Protease Inhibitor Cocktail 1
(-80

C, aliquots)
500 mM
20%
200 ul
200 ul
100 ul
20 ul
200 ul
100mM
4%
10%
2%
50 mM
2%
5%
1%
EDTA (pH 8.0) (RT)
NaF (RT)
Beta-glycerophosphate (RT)
Sodium Orthovanadate 2
(-80

C, aliquots)
Sodium Pyrophosphate 3
(4

C)
500mM
500mM
250mM
200mM
100mM
20 ul
100 ul
40 ul
20 ul
100 ul
10mM
50mM
10mM
4mM
10mM
5mM
25mM
5mM
2mM
5mM
Total Vol 1000 ul
1 Protease Inhibitor Cocktail - dissolve one Complete, Mini Tablet (Cat No. 1836153) in 1ml of ddH
2
0
(Roche) - store cocktail as 100-200 ul aliquots at -80

C
- stable for at least 6 months
2 Sodium Orthovanadate - stable at -80

C for at least 6 months
- needs to be "activated" before use (see below)
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