Arkin JCA PNPAssay

The alkaline phosphatase assay is analogous to a Miller assay for LacZ activity. 

 SOLUTIONS NEEDED:
 Tris Buffer:  100 mM Tris, pH 8.0
 SDS solution:  0.1% SDS
 Chloroform
 PNP solution:  Freshly dissolve a 5 mg Sigma S0942 PNP tablet in 5 mL Tris Buffer
  1. Add 100 uL of culture to 900 uL of Tris buffer.
  2. Add 10 uL of chloroform, 10 uL of SDS solution
  3. Vortext 10 sec.
  4. Add 100 uL of PNP solution
  5. Incubate at 37 about 10 min. (record the time), or until yellow color emerges from positive control
  6. Centrifuge out cell debris
  7. Measure A_420
  8. Activity can be expressed in Miller-like units using the dilution factor of the cells, the time incubated, and the absorbance values