If we are taking 5 values at every 15 seconds of interval then the OD value is going to increase or decrease every time or increased for some enzyme and decreased for some other enzymes?
Whether the OD increases or decreases depends on the nature of the assay.
Sir, I want to just ask that while we are taking the OD values of a sample at every 15 second then the OD value will goes like 1.2331, 1.2342,1.2356, 1.2367,1.2379 or it will be like1.2379,1.2367, 1.2356, 1.2342, 1.2331. @Adam B Shapiro
Hi arun,
That depends on what you are tracing in the beginning either the consumed substrate or the appeared product every case has its own discussion depending on the nature and condition of your assay.
I have taken 200 mg leaf and crushed in 3 ml of phosphate buffer then centrifuged it. After it, I made aliquots of supernatant in 5 tubes( each containing 200microL supernatant). Then I took 150microL of the crude enzyme from one tube in a cuvette and added 3 ml of the reaction solution (4.65 μl 30% H2O2, 3 ml 100 mM
PBS pH 7.0). and then the OD was taken. Reaction solution with 50 μl 100 mM PBS (pH 7.8) serves as a reference. @Adam B Shapiro @Abderahmane Linani
You are describing a peroxidase assay, I assume, and measuring the absorbance of peroxide at some ultraviolet wavelength? If this is the case, then the absorbance should decrease with time as the peroxide is consumed.
However, there may be a confounding effect, which is bleaching of the organic matter in the crude enzyme extract by the peroxide, which could also cause the absorbance to decrease. You need a negative control, which would be to include a substance that inhibits the peroxidase activity.
I suggest you obtain a peroxidase assay kit, if you can, that measures absorbance increase in the visible range. Many such kits are available.
Sir this protocol was for measuring catalase activity and for peroxidase, it was different.
What wavelength were you monitoring? What substance was being monitored at this wavelength?
If I get you correctly, the OD should be decreasing in this case, you probably use wavelength in the ultraviolet range (240nm).
I suggest seeing these previous RG questions that maybe help you with the advice of Pr.ADAM B.
1- https://www.researchgate.net/post/What_is_the_wavelength_for_the_measurement_of_catalase_activity_in_algae
2- https://www.researchgate.net/post/what_is_the_better_method_for_assay_of_peroxidase_in_plant_leaf
Dear, Catalase dissociates hydrogen peroxide, therefore, OD at 240 mm decreases. Peroxidase requires hydrogen peroxide for converting substrate to product. Check the concentration of hydrogen peroxide in reaction mixture. While assaying in UV region the reaction mixture solution should be very clear, if not, the time scan would show ups and down. But easy to perform peroxidase assay as the product is coloured.
yes, the OD will decrease in the case of catalase, but what about others like peroxidase assay, SOD assay.
Dear, How can you assay all the three enzymes assay in a experiment. Normally three enzymes are assayed seperately. Each enzyme catalyse different substrate. If you know specific activity of each enzyme you can determine ABS change. The ABS change should be less than one for accuracy.
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