If I understand your question, your problem is that one of the substrates you use for your enzyme reaction is contaminated with pyrophosphate (PPi), which is also the product of your enzyme that you wish to monitor. The problem, then, is how to remove the PPi contaminant from the substrate. My 1st choice would be anion-exchange chromatography. PPi should bind pretty well to the resin at neutral or slightly basic pH because of the strong negative charge (-4) on PPi. If the substrate is less highly charged (dihydropteroate is a mono-carboxylic acid with charge -1), you should be able to elute it from the resin at a lower salt concentration than the PPi.

I have tried anion exchange and although it helped, the readings are still too high. If I were trying to separate dihydropteroate, that would be easier. First of all, the proposed product resembles dihydropteroate, but it's not. We are still at the assay optimization stage not product identification or separation from reactants. Before we even start the assay, we have to synthesize RFAP, the second proposed substrate, which is contaminated with pyrophosphate/possibly phosphate. And this one has a -2 charge on it and co-elutes with the pyrophosphate, my question is are there any other stronger columns that may cause better separation and how can we go about using them?

Here's another idea. Convert all the PPi contaminant to Pi using inorganic pyrophosphatase. Then use purine nucleoside phosphorylase + guanosine, inosine, or adenosine to consume the phosphate, leaving guanine, hypoxanthine, or adenine, respectively, and ribose phosphate as, hopefully, inert contaminants. Remove the enzyme with a centrifugal ultrafiltration column. All the reagents are available commercially.

Dear all,

I found this question quite interesting.

I would like to use the Malachite Green Phosphate Detection Kit (in my case from R&D Systems) and I would like to use a commercial buffer. Does any of you know a good one?

In some papers it is used MgCl2, NaCl, KCl, glucose and HEPES as a reaction buffer. I guess ddH20 is the diluent. In my hands the usage of HEPES alone did not work.

Thanks a lot in advance!

Anna

If there is a buffer that has been commonly used by other researchers for this enzyme, you can start with that.

If not, the buffer should be optimized for the particular enzyme you are using. The optimization can include choosing the identity and concentration of buffering compound, the pH, the identity and concentration of salt, the Mg2+ concentration (ATPases require Mg2+ for activity), the reducing agent identity and concentration if needed, the detergent identity and concentration if needed, and a stabilizing excipient identity and concentration if needed.

Prepare concentrated stock solutions of all the ingredients you plan to test, and prepare buffer solutions by mixing them together and diluting with Milli-Q water to get the desired final concentrations. This gives you the flexibility to try many different combinations.

You also need to consider the substrates (one of which is ATP), and their concentrations.

Dear Adam,

Thanks a lot for your detailed reply!

Indeed I just tried dH2O and 2 other buffers that have been reported to be used for my application in this kit from another company as reaction buffers. I thought it could work, but it did not. I explain what happened. I performed a calibration curve with the different reagents and in some wells (100 uM phosphate standard dilution in the 3 different reactions buffers, and in 50 uM in the 2 other buffers) there was a green precipitation, which may indicate phosphate contamination. Moreover there was no difference in absorbance between the different concentrations in the calibration curve. Technical service from the kits company said I should try to interrogate the interference of every single reagent, they did for some and was ok, but not sure what happened when they are mixed.

At this point I do not know if it is better just to buy the other kit from the other company and try because the procedure is slightly different, or try to optimize this kit. The kit I am using is from R&D Systems, and the kit from the publication is BioAssay Systems.

Here goes the composition of the 2 reactions buffers I used:

Reaction buffer: 2 mM MgCl2x6H20, 120 mM NaCl, 5 mM KCl, 10 mM Glucose, 20 mM HEPES, 2 mM CaCl2. pH 7.4

Reaction buffer: 1 mM MgCl2x6H2O, 125 mM NaCl, 5 mM KCl, 5.6 mM glucose, 20 mM HEPES, 1.8 mM CaCl2, pH 7.4

Thanks a lot in advance!!!

What is the source of your water, and what containers are you using? Phosphate contamination could be coming from impure water. If you are using glass containers to prepare or store any of the reagents, the phosphate contamination could be coming from the detergent used to clean the glassware. It is best to use only new plastic containers, but if you must use glassware, it should be rinsed very thoroughly with double-distilled or Milli-Q water.

I was using glass containers to prepare my stock solutions of reagents, and the water is also in glass containers. I will change that. Thanks Adam!

Hi Anna,

I am dealing with the same problem with the kit from R&D Systems. My buffers are slightly different but same as you described, there is precipitation in my standard dilutions and no difference in absorbance between the different dilutions.

I was wondering if you could solve this problem or if you have any advice for troubleshoot this.

Hello Cinthia,

in my case the problem were the glass containers. I changed to new plastic containers which I am only using once, and everything is working fine. I guess it has nothing to interfere, but I am also working under sterile conditions (not all the components of my buffers are sterile because they have been opened before at my bench).

I am glad you solved it. Thank you so much Anna!