Typical reaction protocol for lambda protein phosphatase

Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate. For example, 100 units of Lambda Protein Phosphatase remove ~100% of phosphates (0.5 nmol) in phosphorylated myelin basic protein (phospho-MyBP, 18.5 kDa) in 30 minutes in a 50 µl reaction.

  1. Combine protein sample and H2O (if necessary) to a total volume of 40 µl
  2. Add 5 μL of 10X NEBuffer for Protein MetalloPhosphatases (PMP) and 5 μL of 10 mM MnCl2 to make
    a total reaction volume of 50 µl.
  3. Add 1 µl of Lambda Protein Phosphatase.
  4. Incubate at 30°C for 30 minutes.