I am trying to measure the amount of inorganic phosphate using the malachite green assay (Carter SG and Karl DW. 1982. J Biochemical Biophysical Methods). They use a sodium molybdate solution instead of an ammonium molybdate solution (which appears to be more commonly used nowadays). Moreover, after preparing the malachite green dye, the final colour is a very dark green. With such a dark colour, I am having my doubts of whether I will at all be able to measure inorganic phosphate in my solutions. Tips on troubleshooting would be great. Detailed protocols of how to perform the assay for proteins like ATPases (dyneins) would be of great help.
Phosphate contamination of glassware used in the preparation of the reagent must be avoided. Wash all glassware thoroughly with Milli-Q water. Make sure all of the reagents used in the preparation are also free of phosphate.
After preparing the reagent, filter it through a vacuum filtration funnel to remove precipitated dye that inevitably forms. The filtrate should be brownish in color. Store the reagent in the dark.
Dear Askash
as Adam suggest you need to avoid phospate contamination.
Use only milliQ water for buffer preparation. Subtract all you values for a blank sample.
Attached you can find a datashet of malachite green assay commercialized for R&D sistem where you can find some details about the protocol.
I used it for estimation the amount of phospate generate for hydrolisis of UDP form a clostidium glicosil tranferase and it works quite well.
good luck
Manuele
Here's another detailed protocol with home-made reagents. Simple, reproducible.
Can you please tell me which type of polyvinyl alcohol is used in malachite green assay. the link is as follows:
https://www.sigmaaldrich.com/catalog/search?term=polyvinyl+alcohol&interface=All&N=0&mode=match%20partialmax&lang=ko®ion=KR&focus=product
The simple protocol I added before does not require any polyvinyl alcohol.
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