I am trying to measure the alkaline phosphatase activity for algal cells as a response to phosphorus deficit. For the methodology I have checked various articles that deals with a similar topic. In short, the process include mixing a sample (algal cell suspension) with pNPP substrate and Tris-HCl buffer, incubating it, adding a solution to terminate the reaction, filtration and light absorbance measurement. However, in every article I have read the volume and concentration of sample, pNPP substrate, buffer and stop solution as well as the incubation time and temperature are different.
My question here is how to determine the the right volume and concentration for the sample and reaction solutions and the incubation time and temperature? Is it even possible to theoretically determine that? Or is it case-specific, and I need to do a series of tests to find the right recipe for my case?
@ Aigars, in general 0.025M p-nitrophenyl phosphate solution is very effective in universal buffer. The optimum incubation time is 1h at 37 degree Celsius. It is not case specific, I hope you may get consistent results.
Each enzyme has an optimal pH, ionic strength, and temperature. Each enzyme has its characteristic substrate Km, which also depends on the conditions. However, for your purpose, it is not necessary to optimize every parameter. Since you are only interested in the effect of growing the cells in a low phosphorus condition on the amount of alkaline phosphatase activity, you can simply choose a reasonable set of assay conditions from the literature and use it to compare the AP activities from cells grown under different conditions.
Be sure that you are measuring the reaction rates correctly. You should make time-based measurements to be sure you are measuring the initial rate of the reaction. You may have to try various dilutions of the cell extracts to find a dilution that allows you to measure the initial rate accurately.
The reaction volume will be determined by your equipment. If you are using a spectrophotometer and cuvette, the reaction volume must be large enough that the light beam passes entirely through the sample, not through the air above it. There must not be any air bubbles in the light path either.
The pNPP assay can be run in a continuous fashion by measuring the increase in absorbance repeatedly in the same sample while the reaction is occurring. No quenching and filtration steps are necessary. This makes it much easier to measure initial rates.
Dear, Rinse algae with PBS, pH 7 or check the pH of crushed algae solution and assay it. It may work. All the best.
- Badges
- Science method