Any advice or views on a biochem assay problem most welcome

[Oliver Wood]

I'm having a spot of bother with a monooxygenase assay for a certain mosquito larvae. When i add the hydrogen peroxide the colour change is almost immediate, but it starts to fade after a while, and the colour is pretty much gone by the time i'm meant to read the plate.

 

This is what i've been doing:

 

20ul of homogenate in duplicate

80ul of 0.1g 3,3',5,5' Tetramethyl Benzidine (crusher) dissolved with 5ml methanol, plus 15ml of 0.25M sodium acetae buffer pH 5.0 to each well.

25ul of 3% hygrogen peroxide to each well

Read at 1 and 2hrs at 650nm endpoint.

 

I've repeatedly checked my buffer pH's and made sure the mixtures in each well are all stirred up.

 

Any suggestions are greatly welcome...i can't really afford to have to toss more data...running out of specimens.

 

Thanks

Cheers

Ol

[zule]

Why don’t start reading at the moment you add the hydrogen peroxide? Or still better, after adding the monooxygenase.

Then you read the plate until the absorbance stars to decrease and choose the maximum value to make the calculations.

[Oliver Wood]

yeah, i was hoping that wasn't going to be the advice. was hoping that this was a common problem with the titration.

 

I suppose what i could do is monitor the curves and when they peak or level off take that reading.

 

Pitty though, means i can't do my other assays in the wait time

 

But thanks for your imput, i'll certainly investigate the opions it offers.

 

I'm going to go to the insectary now and harvest some baselines to grind for tomorrow's attempt. (bugger this is going to be a terrible week!!!)

 

 

Cheers

[Gyrase]

2 hours is too long, Tetramethylbenzidine is less stable comparing to other chromogenes like 4-aminoantipyrine.