Hello ,
i am doing catalase assay for plant extract. The reaction mixture contains (2.5ml) contains 1ml of 50mMphosphate buffer(pH 7.0) , 0.5ml of 0.5mM H2O2 and 1ml enzyme extract of concentration 200ug/ml.The absorbance is read at 240nm. I am getting OD more than 1 and in uneven pattern also.Is it because my protein got denatured or because of some other reason.
Kindly suggest me.
Thanking you in anticipation.
Dear
The high absorption could be due an instrumental error, try again with different compounds and low concentration, if you get reasonable absorption that means it's from your compounds you used in assay. Check your solutions concentrations and make sure that your solutions are not turbid.
During plant extract preparation did you use PVPP or PVP to reduce contamination from polyphenols, phenolics, pigments, chlorophylls etc?
Uneven pattern could be a bad spectrophotometer (test it using only buffer and monitoring for 1-2 hrs) for consistency. Uneven by how much units? If too much, then due to oxidizing pigments or denaturing proteins etc. - easily so.
Also, use a negative control blank using everything (including your plant extract!) except H2O2, then you would monitor only H2O2 after doing 'blank'!
Thanks,
Dear Colleague
Your problem here definitely could be as a result of either a pre-analytica issue which could be from reagent preparation, dilution, sample preparation, dilutions, contamination from any sources;( remember you are measuring an enzyme, so check out for enzyme activators) ; or analytical issues such as: wrong pipetting, poorly calibrated system (pipettes, specs, balances etc) interference from any sources and standard issues. Once you troubleshoot and fix any or all of these likely issues, your analysis will come out perfect.
Good luck.
Troubleshoot as advised by other colleagues. Also, you may endeavor to sieve your extract through muslin clothe or spin it in a centrifuge in order to reduce turbidity.
Best wishes!
I suggest you carry out a serial dilution of your extract and probably prepare a fresh analytical solvent solution. Thank you.
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