Hi all researchers,
I want to perform PTP1B experiments. Previously I performed this experiment and analyzed data but all the values were negative. Someone told me that my pipetting is not good, and that the enzyme and substrate nature is very sensitive. But I paid attention to both and performed experiments many times. Unfortunately, the results once again were in negative values. Please suggest a proper way to handle and pipet the sample, enzyme, substrate etc. to a 96 well plate.
What is the nature of the assay you are using? How large are the negative values? What is your negative control? Have you done a standard curve with the product of the reaction?
Did you use something like the pNPP assay (the method is unclear from your question)? Then the method is quite easy and not sensitive to minute errors in pipetting. Check the pH of the buffer use (the PTP activity is pH sensitive) and check for the presence of any denaturing agents or reactive oxide species in your buffer since the enzyme needs to be properly folded to be active.
- Did you use a second wavelength for correction, set to the isosbestic point (the wavelength where the absorbance is independent of concentration)? This can be used to account for imperfections in the plate bottom, like scratches, schlieren, finger prints...
- In addition, you need to use a proper negative control (enzyme replaced by buffer).
- In some assays absorbance is actually reduced, as the coloured substrate is converted into non-coloured product. In such cases, the blank needs to be without substrate.
- It is good practice to do assays in one order (say, increasing substrate concentration) and then repeat them (on the same day with the same batch of chemicals) in reverse order. Use different symbols to plot both data sets in the same graph. Any decomposition of substrate or enzyme during the day will become immediately apparent.
- Have somebody show you how to use pipettes properly, and then do 3 important things:
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