I'm using phenol red for measuring the activity of MnP enzyme. From the literature I got the reaction mixture contained 25 mM lactate, 0.1 mM MnSO4, 1 mg/ml of bovine serum albumin , 0.1 mg/ml of phenol red, and 0.5 ml of culture filtrate in 20 mM sodium succinate buffer (pH 4.5) in a total volume of 1 ml. The reaction was initiated by the addition of H2O2 to final concentration of 0.1 mM and was stopped after 1 min with 50 μl of 10% NaOH, and A610 was measured. Control is made with omitting MnSO4 from reaction mixture. Enzyme activity = (A610 for test assay) – (A610 for control assay).
What is the blank for this assay? I don't know the blank so I'm using distilled water as a blank, and it turned out the value of enzyme activity negative, because control always higher than test..
Does my blank is false or my reaction mixture?
Thank you for your very kind attention
No.. it says that the enzyme ativity is A610test-A610control. The control's color is just the same with the test tube.. Right now, I'm using blank with culture supernatant is excluded from the mixture. But it results in absorbance of the test is 0.007, and so on..
Dear Miss Ira,
In fact you assay here is a decolorization assay...that's means that you measured at 610 nm the loss of the red color, then it's normal that when you have more MnP activity you have less absorbance than the control. Consequently the mistake here is the formulae: you should use Abs (control)- Abs(Assay) = Abs relative to MnP activity.
Regarding the blank, I don't think that the use of distilled water influence a lot the absorbance at 610 nm. However, if your culture filtrate have any colour, you should use a blank containing 25 mM lactate, 0.1 mM MnSO4, 1 mg/ml of bovine serum albumin and 0.5 ml of culture filtrate in 20 mM sodium succinate buffer (pH 4.5) omitting 0.1 mg/ml of phenol red. With this blank you should not have any doubt.
Another point to consider: I suggest you to extend the time reaction to 3 min for example, because if you have little activity your reaction will be very slow and you will not detect its.
As a control, you might try using the same medium as the culture medium, but that has not been used for a culture.
Dear Sir Said Galai & Adam,
Thank you very much for your answer.. I'll try it using your method :)
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