Hi all,
I am doing an Alkaline Phosphatase activity assay using PPi as substrate. I am using PiBlue Kit to measure amount of Pi generated at 620nm.
I am getting high OD values in my no substrate control ( no, PPi) than my enzyme inactivated (4N H2SO4) control. Basically, there should be minor difference between no substrate control OD and enzyme inactivated (4N H2SO4) control. This is resulting in failing of my assay?
Why do you all think it is happening?
Thanks,
Arshed
Arshed Nazmi You have not menioned whether you are dealing with pure enzyme or crude extract. If you are using crude extract, then it would be better to filter your enzyme through 3 kD or 5 kD column and then use it for estimation of activity. Then you may not see the high reading in no substrate blank.
If you are dealing with pure enzyme, please comment.
Please share the reaction mixture, and assay procedure, it would be easy to comment on that.
The simplest explanation could be that you have some contaminant phosphoester in your reaction mixture, which gets hydrolyzed in the presence of the phosphatase (but not, obviously, in the presence of inactivated enzyme). Try wether changing buffer or using a different source of MilliQ water gives you different results...
Phosphatase activity assay is very sensitive. The problem might be arisen from phosphate contamination. To avoid such contamination, it's better to wash all glassware thoroughly with sterilized distilled water. All of the reagents used in the preparation should be phosphate-free and stored in dark place (or raping with Aluminium foil paper).
@Rehenuma,
thanks, for your suggestion.
How can I ensure my reagents and glassware are phosphate free? Is there a way to do that?
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