I am not sure how much volume of these two chemicals should be added while following the said protocol. Advice on it is appreciated.
Hello ali, in general the amount of extracted soluble proteins depends on the type of plant and buffer used. If you are using just the Tris buffer with protease inhibitors only, i will recommend you to use 1:2 extraction buffer (1gm of leaves in 2 ml) buffer. In case you are adding sds or detergents like Triton-X 100 or Tween-20 in your extraction buffer, the amount of extracted proteins would be much higher. In that case you can use 1:5 extraction buffer (1gm leaves in 5 ml extraction buffer). Different protease inhibitors have different effective conc., so it would be better if you could explain which protease inhibitor you are using. Are you using PMSF of some cocktail?
The final concentration of PMSF is 1 mM recommended as said Yang Liu.
Generally, I prepared 0.1 M PMSF stock (Prepare just before use; add 1.7419 mg in 100 μL of methanol) and added 100 μL PMSF in 10 mL buffer. You may calculate according to your required volume.
- Badges
- Science method