I am trying to optimize the Enzchek Phosphate Assay kit (Thermo Fischer Scientific) for a reaction volume of 100uL, while the kit has been designed to work in 1mL. I calculated the reagents according to 100uL and set up the reaction to getting a standard curve for reaction volume of 100 uL, but there has been a lack of linearity in 100uL reaction mixture as opposed to a perfectly linear graph when the standard reaction was set for 1mL.
My goal is to optimize the reaction for 100uL so that I can run a phosphatase assay for the enzyme and substrate of my interest.
If anybody has used this kit and worked for a smaller volume, then please do suggest the needful.
Thanks in Advance
Yes, I am using that kit all the time! I suppose you are having trouble getting a good signal/noise ratio, and your readings fluctuate constantly?
You can follow the protocol exactly to the letter, but just cutting the volume by a factor of 10 - as you are doing already.
You are not using cuvettes, but rather a microplate reader, correct? If so, you can not expect to get a good reading using a standard 96-well plate. It is absolutely critical that you use UV-transparent microplates, as they do not cause nearly as much background noise. You can either use quartz or purpose-made polystyrene plates, and it also doesn't matter if they are black-rimmed with transparent bottoms or completely transparent.
Good luck with the kit.
Hi Sneha,
I am not using the assay you mentioned but I have worked with phosphate and phosphatase assays a lot. To measure the released orthophosphate very sensitively I found the PiColorLockGold assay from Novusbio (Cat#303-0030) working very reliably. I always used exactly 100uL of my reaction, which I added to 25uL kit detection reagent (2 components, very easy) in a 96well plate (absorbance at 635nm). You get very little deviation between technical replica and the standard curve of phosphate is straight from 0.125nmol to 4nmol phosphate per 100uL sample volume. There is also a stabilizer reagent in the kit which helps if your phosphatase substrate is labile. Let me know if you have more questions.
Best, Felix.
Sneha Roy I use the 20X buffer that comes with the kit.
With that being said, are you already using UV transparent plates for these measurements? How are you measuring your signal, using a plate reader or do you just use smaller cuvettes with a 100ul reaction volume and measure them that way? It is a bit easier to troubleshoot when the experimental setup is clear
Michael Adams, Thanks for your response. So, firstly I wasn't using the UV transparent plate, however, I have placed an order for those now. I am using a 96 well-plate for the assay. The issue is, by the time I start observing the absorbance, it reaches saturation (Abs ~0.2 at 360nm and doesn't change at all while I am recording the kinetics).
Here is my reaction protocol:
MESG: 200uM, PNP: 1U, Buffer: the one I use for my sample (not the 20X), Test enzyme: 0.2nM, Test substrate= 200nM (as well as other varying conc.).
I add the peptide in the end and then start monitoring the absorbance for 1h at an interval of 2min. But I believe the reaction already reaches saturation by the time I start recording.
Is there something I am doing incorrectly except using the sample and not the 20X reaction buffer? Also, the absorbance is high, according to the datasheet given with the kit.
Thanks in advance for helping.
Just to be very clear: In your protocol, you are adding purine nucleoside phosphorylase (PNP) but you are not adding inorganic pyrophosphatase (PP)? You need to add them both, or you will not detect any signal. You should always have one condition where you add just nucleotide without your experimental enzyme and the PNP and PP to do baselining with, but it is important you add both PNP and PP to your reactions including your own experimental enzyme
It depends on what buffer you are using - If you have something between 2 and 10 mM MgCl2 in your buffer, I don't see how this would cause an issue since the kit is basically just a tris-buffered solution with Magnesium and sodium azide.
So the saturation you are talking about is with your enzyme in question, not the standard curve? Because in the curve, your readout depends on it having reached saturation, and you get the line fit because you are titrating increasing amounts of reagent into the wells.
For your enzyme, it is very possible that the reaction catalyses quite quickly, especially if you add high micro molar concentrations. You could try a serial dilution of your enzyme and see if you can find a concentration at which you can capture the reaction taking place over the course of an extended period of time - the rate of catalysis varies greatly between enzymes, so this is one more avenue you can consider.
If you had a clear signal before using UV transparent plates, you would have most likely picked up that signal already using the standard plates, the transparent plates will only improve the signal/noise ratio a lot.
Sure, but my protocol is really simply following the instructions in the kit, and adding 2uM of my protein (final concentration) and 2mM ATP as a pyrophosphate source to the reaction. Look at the section titled 'Inorganic Pyrophosphate Released Continuously by Enzymatic Reaction', and divide all the numbers by 10.
Felix Nitschke please correct me if I'm wrong but the difference between the products is that EnzChek allows the continuous detection of phosphate (for progress curve measurements) while the PiColorLock needs a resting time before one can make a reading. So far, EnzChek or other kits using MESG substrate are the only ones I've found for kinetic measurements.
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