Jacobs:Protocol Alkaline Phosphatase Assay

Materials

 * -20 freezer
 * RIPA lysis kit
 * PMSF solution
 * Protease inhibitor cocktail
 * Alkaline Phosphatase Fluorometric Assay Kit (Sigma Aldrich - APF; Abcam - ab83371)
 * Control enzyme
 * Substrate


 * Fridge
 * Buffers from APF kit


 * Miscellaneous
 * 96 well plate
 * 1.5 ml microcentrifuge tubes
 * Pipette, tips

Preparation

 * 1) Buffer
 * 2) * 20 ul of dilution buffer and 160 ul of fluorescence buffer is needed for each sample
 * 3) * Prepare enough of the working buffer for the standards and unknowns
 * 4) Standards
 * 5) * Dilute stock control enzyme in water. 1:100 to 1:6400 has worked well. This correlates to 1 to 0.015625 ug/ml alkaline phosphatase.
 * 6) Samples
 * 7) * Dilute samples to ensure the samples fall within the standard curve. At least a 1:2 if not also a 1:10 dilution is recommended. Alternatively, you can aim for between 1 and 10 ug of protein per 20 ul.

Assay

 * 1) Prepare the wells by adding 20 ul of each standard or sample in triplicate.
 * 2) Add 180 ul of the working buffer to each well.
 * 3) Add 1 ul of the substrate to each well. Do this quickly as the substrate will immediately be converted.
 * 4) Measure fluorescence in the plate reader at 330 nm excitation and 440 nm.  Generate a standard curve of FSU vs. alkaline phosphatase in ug/ml.