Hello, I am planning an experiment to measure enzimatic activity, and i will take sample every 24hrs. The only problem is that substrate is non soluble... nevertheless, enzyme can still access to it. My idea is to keep the reaction on movement, but i would like any suggestion on how to lessen error when taking the sample, because last time i tried doing separate reactions in different tubes for each measure (e.g. i had 3 different tubes for T0, 3 dif. tubes for T2, and so on), but replicates wheren't as much as similiar as I expected them to be. My first thought was to make a reaction stock (on triplicate), and take a volume of it each time, but I believe that since the subtrate isnt soluble, each time I take a sample, subtrate might vary, and i will still get error...
Is there an alternative for this type of experiments?
If not, which way is better: a stock reaction or multiple reactions?
Thank you in advanced for reading.
I think the most important point is to make sure the insoluble substrate is well-suspended at all times. Given that, I would expect to get the same results whether using a single volume sampled multiple times, or separate volumes for each time point.
Since enzymes can lose activity if mixed too vigorously, you have to find the optimal compromise between keeping the substrate suspended and causing damage to the enzyme from excessively vigorous mixing.
Oxidation of the enzyme (and substrate?) may also be a problem for such a long-term experiment. It might be helpful to flush the container with nitrogen to reduce oxidation.
June 22, 2023
Dear Valentina,
You received several excellent suggestions from Dr. Shapiro.
I don't know what your substrate is, but, if you haven't already attempted this, I would try to macerate the substrate. You could do this with the dry material, or a buffered suspension of the solid. There are various apparati that might work for this, e.g., Waring blender, grinder, mortar & pestle, etc. The idea is to convert the solid substrate into tiny particles that, on addition of your buffer, produce as uniform a suspension as possible. Converting the solid material into small particles will help keep the material in suspension and, more importantly, will greatly magnify the surface area of the substrate for contact w/ your enzyme and increase the rate of enzymolysis. Flushing the suspension w/ N2 prior to addition of the enzyme would prevent or minimize oxidation.
One way to gently mix the enzyme-substrate suspension would be to put -the mixture in a tube and fasten it horizontally on a rocker-type mixing platform. This would allow you to gently rock the tube back and forth at a pre-determined rate, keeping the particles in suspension for action by the enzyme without damaging it. At predetermined intervals, you could withdraw samples from the tube to measure the reaction product.
I hope this information helps you.
Bill Colonna Iowa State University, Ames, Iowa, USA [email protected]
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