Recommendation for use: Equilibrate reagent to room temperature before use.Following final binding reaction with an ALP labeled probe, wash the membrane in Tris-buffered saline or Tris/HCl containing 0.1% Tween 20. Do not use phosphate buffer for washing since this will inhibit ALP.Incubate the membrane in Agrisera BCIP/NBT ALP Substrate solution, protected from light, for 5-15 minutes. Make sure the membrane is completely covered in solution. Depending on enzyme activity longer incubations may be necessary. If the concentration of ALP probe is too high, color might develop almost immediately, and the formazan deposit can flake of the membrane. High concentration of ALP probe can also result in formazan deposits forming a thin line around the band or dot, with no deposit in the center. This requires further dilution of the ALP probe.Wash the membrane with deionized water to stop the enzyme reaction.Dry the membrane and store at room temperature protected from light. Purple bands or dots will be visible at the sites of ALP activity. In case of excessive background staining try increasing the number of washes of the washing time since this indicates an incomplete removal of non-bound ALP from the membrane.