Biochemical Techniques and Instrumentation

ALP substrate

• The substrate for HRP is hydrogen peroxide. Cleavage of hydrogen peroxide is coupled to oxidation of a hydrogen donor which changes colour during reaction.
• This reaction can be stopped by the addition of 0.75 M NaOH. (3,3’,5,5’-tetramethylbenzidine) is added as substrate to each well, incubate for 15-30 min, and add equal volume of stopping solution (2 M H₂SO₄) and read the optical density at 450 nm.
• TMB
• If OPD (o-phenylenediamine dihydrochloride) is used as subtrate the end product is measured at 492 nm. Be aware that the substrate is light sensitive, so keep and store it in the dark.
• If ABTS (2,2’-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt is used as substrate the end product is green and the optical density is measured at 416 nm.
• Some enzyme substrates are considered hazardous (potential carcinogens), therefore always handle with care and wear gloves.
• Dispense 100 μ l (or 50 μ l) of the substrate solution per well with a multichannel pipet or a multipipet. For most applications pNPP (p-Nitrophenyl-phosphate) is the most widely used substrate. The yellow color of nitrophenol can be measured at 405 nm after 15-30 min incubation at room temperature.
• After sufficient color development add 100 μ l of stop solution to the wells.
• Read the absorbance or optical density of each well with a plate reader.

Analysis of data

Prepare a standard curve from the data produced from the serial dilutions with concentration on the X axis (log scale) vs absorbance on the Y axis (linear). Interpolate the concentration of the sample from this standard curve.