I tried to set up a malachite green assay to measure the ATPase activity of my protein and basically it seems to work but there are a few problems.
First, termination of the reaction. I could not find a detailed protocol but only what is described in a number of publications (which leaves a lot of space for interpretation of the method!!). Most descriptions said that people terminated the reaction by adding the malachite green reagent (MGR - 1:1:2:2 0.572% ammonium molybdate/6N HCl:0.232% ployvinyl alcohol:0.0812% malachite green:ddH2O), some people said they deactivated the protein with TCA. After adding the MGR I wait ~30min and then do the measurement at OD630. However, the reaction does not stop because the samples get greener and greener over time. I doubt that it's just ATP that breaks down because the "ATP only" control gives a very low OD reading and does not change over time. I also could not find anything on how people precipitate the protein before adding the MGR to stop the reaction. By adding TCA to a final conc of 10%?! And then, spinning it down and taking only the supernatent?
And second, the sensitivity of the assay. I found a whole bunch of publications were people using this assay describe the activity of their protein in the nm range (1-40ish nm). When I do my standard curve using KH2PO4, the first kind of reliable reading I get with about 100nm Pi (OD630 = ~0.04). Everything below is not significantly higher than the blank. Am I doing something wrong or is there a way to increase the sensitivity of the assay?
I also would be very thankful if someone has a protocol he/she is using in the lab and that has some details.
Hi Marco,
I can send you our detailed protocol (please give me your email address). We do not inactivate the proteins, as the assay solution contains 1 N HCl (which sould give a pH of 0 and inactivate all proteins). Our assay solution contains 1 mM malachite green and 8.5 mM ammonium molybdate supplemented with 0.1 % Triton X-100 shortly before use. The sample (50 µl) is incubated with 800 µl of the assay solution for 1 min at RT. After addition of 100 µl of 34% citric acid and an incubation time of 40 min at RT, we read out absorption at 640 nm. We use KH2PO4 (0-200 µM) for the standard curve. The detection limit is 10 µM (in the 50 µl sample, which gives 500 pmol of phosphate as the limit) with our assay. If the A640 readings exceed 1, lower protein concentration. The assay works quite fine and stable in our hands. Good luck, Magdalena
Hi Marco,
I can send you our detailed protocol (please give me your email address). We do not inactivate the proteins, as the assay solution contains 1 N HCl (which sould give a pH of 0 and inactivate all proteins). Our assay solution contains 1 mM malachite green and 8.5 mM ammonium molybdate supplemented with 0.1 % Triton X-100 shortly before use. The sample (50 µl) is incubated with 800 µl of the assay solution for 1 min at RT. After addition of 100 µl of 34% citric acid and an incubation time of 40 min at RT, we read out absorption at 640 nm. We use KH2PO4 (0-200 µM) for the standard curve. The detection limit is 10 µM (in the 50 µl sample, which gives 500 pmol of phosphate as the limit) with our assay. If the A640 readings exceed 1, lower protein concentration. The assay works quite fine and stable in our hands. Good luck, Magdalena
Hi, I have done a lot of ATPase assays over the years and found the regenerating assay coupled to NADH to be the best in terms of both real--time assay and real results without too much of a background (there is a good protocol in an older paper of mine (McMillan et al 2007, or dig out the ref I used if you like). However, I am aware certain enzymes do not behave for whatever reason so the inorganic phophate assay is similar to the one described above that you can find in my A1Ao ATP synthase paper or of course who I referred to! (note that you absolutely MUST do good controls with any Pi assay on the day - its critical!).
On the note of your assay, Pi assays usually must be measure immediately and you must be accurate about keeping your timing. Also, check the background of your ATP if you have not already done so....you may have significant Pi in there already. The sodium-ATP is best for stability if Na is not part of your experimental question.
Dear Marco,
we use the method described in Methods Enzymol, 1990;192:608-16. Isolation of goblet cell apical membrane from tobacco hornworm midgut and purification of its vacuolar-type ATPase. Wieczorek et al. which was developed in our lab. In this protcol the enzym activity is first stopped by rapid freezing of the eppendorftube with the sample in liquid nitrogen. For phosphat determination the sample is thawed in the presence of TCA, which effectivly denaturate the enzym. With this protocol we reach nmol sensitivity.
Try stopping the reaction with 200 microlitres of 5% sodium dodecyl sulphate (SDS). Since the ATPase is membrane-bound, the SDS will disrupt the lipid bilayer and stop the enzyme activity. I find this a better procedure than the precipitation with TCA.
Dear Clement,
FYI:
In my experience for most ATP synthases the SDS treatment may not work, and certainly does not work by a mechanism of bilayer disruption. The reason why is the the A1/F1 parts of the holoenzyme are able to hydrolyze ATP without the membrane-bound Fo/Ao portion attached. Which is incidentally the reason why ACMA quenching and synthesis activity are the standard 'holy grail' for proof of fully ion-linked enzyme. But then again, this is quite a bit of SDS in there, but I would be very careful of the assumption its all dead....
The comment made by Duncan is very important. Not only malachite green assay also other Pi assays usually must be measured immediately as soon as possible. Even though protein was denatured, ATP is spontaneously hydrolyzing to ADP and Pi by the thermal reaction.
thanks a lot for your responses and your useful help. As mentioned in my initial post, the main problem, I think!, is to stop the reaction because the more time passes by the higher the OD readings of my samples get. And I am pretty sure it is not just the ATP spontaneously hydrolyzing to ADP + Pi because the OD of this control samples does not increase to such an extent as when protein was added. However, I also realized that it takes my standard about 10-15min to stabilize, so I guess that's kind of a bottleneck in terms of timing!?
Anyways, I'll try some of the different suggestions and see what works best for my assay.
so thanks a lot again and all the best
In my experience, ATP hydrolysis by Malachite Green/molybdate reagent is a significant effect causing the absorbance to increase with time. (I use the reagent of Itaya and Ui that also contains 1 M HCl but no PVA.) Therefore, I make the measurement at a specific time after reagent addition, usually 5 min. I'm surprised you don't see it in your no-enzyme control. It would be remarkable if the enzyme retained activity in 1 M HCl. Are you sure you aren't seeing turbidity caused by precipitation of your enzyme? What is the protein concentration? Too much and there will be detectable precipitation. Try a control with enzyme but no Mg2+.
As for the sensitivity, I find that the homemade reagent allows measurements between 2 and 20 micromolar in a microplate format (30 microliter reaction + 30 microliter reagent in a 384-well plate read at A650), i. e. 40 - 400 picomoles.
The Malachite Green assay is much more sensitive that the NAD/NADH absorbance assay and doesn't require a coupling enzyme, but the latter is very useful because it allows for continuous measurements. Its sensitivity can be improved by using NADH fluorescence increase instead of absorbance.
I'd agree with Adam's suggestions about the sensitivity of the assay. I found that reliable measurements are possible down to 2 micromolar.
To answer your initial question, yes, the colour will continue to alter over time. This is why it is so important to run a phosphate ladder in every reaction, and use a standardised protocol.
It is important to note with the malachite green assay that it has a relatively limited dynamic range (usually it is reliable over about one and a half orders of magnitude). However, you can alter the phosphate concentration range over which it is sensitive by modulating the mixture of malachite green, ammonium molybdate, and the acid; and by altering the ratio of detection reagent and test sample. It sounds like your current mix is only sensitive at high phosphate concentrations. I would suggest reading a detailed methodology paper (e.g. Cogan et al., Anal. Biochem., 271, 27-35, 1999).
One other suggestion is to use the Pi Colorlock reagent, from Innova Biosciences. This is an excellent pre-prepared reagent, and includes a chemical that helps prevent lysis of ATP after adding the malachite green reagent. It is expensive, but I have found that it significantly improves the quality of data that we get, so it's worth the money!
Adam: thanks a lot for the info. So I measure some absorbance in my control that contains only ATP (e.g. 2mM ATP, OD630 = 0.05) and it is significantly less compared to the same sample that contains 2ug of my protein (OD630 = 0.42). However, about 45min later the same protein sample has an OD of 1.3 while I did not observe such a drastic change in the control (forgot to measure it in my surprise but there was not a dramatic color change, still just slightly greenish). And this made me wonder because if it was only the ATP breakdown, color changes should have been similar = the control should have turned bright green. At this point it's also not precipitated protein. Eventually there is some (or a lot) turbidity in the samples but not within 2 or 3h.
However, I like your idea of including a protein sample without Mg2+ and will add it to my next experiment
Nicholas: Thanks a lot for the paper reference, that looks really good and I will try their experiment to figure out the optimal malachite green and ammonium molybdate concentrations for my experiments. Regarding the phosphate standard, shouldn't the standard determine how long one has to wait to measure the samples? Means, shouldn't you wait until the color reaction in the standard samples stabilized and choose this timepoint to measure your samples? Because, at least in my case, the standard doesn't change anymore over time (which makes sense because there only a certain amount of phosphate to react with).
Hello Marco,
Most have been answered already. Nevertheless.
The Malachite essay is excellent and very sensitive as mentioned above. Nevertheless it needs to be done with cautious. Pi with Malachite assay is stable for hours (a day without problem).
In contrast Nucleotides such as ATP in acidic condition is degraded and Pi increase significantly over time. For this reason I use to suggest to measure always at the same time for more reliability (25 or 30 min after the addition of the Malachite allowing time for full detection and not too much for increased background) (this does not allow to do less controls in the contrary). In the mean time you need to stop the reactions of your controls in the same time as the assay to avoid problems. S you can not start with the assays and do the blanks later. For example nucleotides can be added right away, then stopped the reaction with Malachite in the same time as the assay and right after add the protein samples to the control as a background of the Pi levels present in the protein sample. Lots of Pi present in the protein sample and also a lot with the nucleotide. SOme compagnies or stock are better then others. Be carefull too as mM concentration of nucleiotides is often a problem with Malachite because of background Pi coming with nucleotides.
Another thing. generally proteins do not need to be precipitated and as mentioned above this acidic condition kills enymes. I never had to do this but if you still need to precipitate them please find the original protocols. Below the 2 original references about Malachite where they did everything (the oldest one the best! not the opposite so start with the oldest one below). No need I think to look at more recent papers. No need to buy kits. Very easy to do.
Good luck with your experiments:
Baykov, A.A., O.A. Evtushenko & S.M. Avaeva. 1988. A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal. Biochem. 171: 266-270.
Geladopoulos, T.P., T.G. Sotiroudis & A.E. Evangelopoulos. 1991. A malachite green colorimetric assay for protein phosphatase activity. Anal. Biochem. 192: 112-116.
I you like our detailed protocole in my lab for nucleotide hydrolysis you are welcome to ask.
Wishes,
Jean
Marco - the issue with how long you leave the samples before reading is that the coloured particles tend to flocculate, especially at the higher concentrations. If you leave your samples long enough, you will see this macroscopically (the sample separates into large suspended deep green particles, and a largely uncoloured liquid). The flocculation doesn't change the colour, but does affect the absorbance, as the flocs scatter light non-specifically.
Of course, depending on the concentration of all your reagents, this may be more or less of a problem. You can always test it by mixing your samples, and reading the absorbance every five minutes for an hour to see what happens. If there's no change in the standard, then there is no problem.
My experience has been that there is enough day-to-day variation in the precise equation of the standard curve to make it the biggest source of error in the experiment if I don't do the standards every time.
From your comment above, it does sound like there is something unusual going on with your sample. One great scientist said that the best science comes not from the experiment that makes you say "Ah, just as I suspected", but the one that makes you say "Huh, that's weird". Don't be concerned - you have noticed the unusual phenomenon, and are tracking down the source of it. That's the first two steps in a potentially important discovery. Good luck!
Hi, Marco. In my experience the dye reagent alone is definately enough to kill the enzyme activity. I can't comment on the ATP degradation issue, as I use the assay on purified lipid phosphatases as opposed to ATPases. Another thing to consider though, does the ATPase you add to the assay have any absorbance alone when dye is added? From my experience any phosphate contaminants in protein preps will affect the assay enhancing absorbance reading. In my experiements, in the negative control after adding the malachite green dye to terminate reactions, I added the enzyme back at the end, thus both enzyme +/- wells have exactly the same contents. Hope that helps.
Actually from my experience, it is very difficult to assay ATPase. This is primarily because as of date there is no suitable inhibitor to stop the enzyme in such a short time and secondly ADP too undergoes hydrolysis naturally. It is therefore, better to use some other phospholipase for the purpose. However, if one wants to assay ATPase then it is better to monitor it by measuring the degradation potential by a suitable Wheatstone bridge.
For me citric acid did the trick ;)
http://www.ncbi.nlm.nih.gov/pubmed/20419410
Dear Marco,
I have personally measured ATP hydrolysis by using some ready-to-use Pi-detecting reagents based on malachite green: PiColorLock (Innova Biosceinces) and Biomol Green (Biomol Research Laboratories). The PiColor comes with a "stabilizer" solution preventing the inevitable increase of the signal with time. For the same reason I used to add 1/10 initial reaction volume (tri)sodium citrate 34% after 5' of incubation with the Biomol Green.
In principle both of the reagents allowed detection of Pi in low nmol range (=Pi concentration in the sample is microM, i.e., using our spectrometer 5microM Pi was the lowest concentration which I used for standard curves).
I recommend you: (i) getting a known ATPase as a control for your protein, (ii) titrating your protein until you get reliable signal, but watch out as with high protein concentrations (microM) it will likely fall out of the solution messing your result.
If you have an access to isotop lab and don't object working with P32 you might try using trace a32P ATP (or g32PATP) in your reaction and resolve the stuff on PEI cellulose (for example CEL 300 PEI from Macherey Nagel) in phosphate buffers (Cashel et al, 1969, J. Chrom. 40, 103-109) and quantitate the spots. This might help you overcome the increase in signal in the colorimetric assays and possibly some precipitation problems. I can also confirm your colorimetric results making them more credible. Your reaction could possibly be stopped upon addition of some 50mM EDTA, formic acid to 50% or just some 10 volumes of phosphate buffer. I don't recommend the TLC approach if your protein requires high concentrations of ATP for its full activity (say 2mM), long incubation times and even though hydrolyzes less than 5-10% of the total ATP in the reaction tube.
Hi Marco Straus. I agree with many answers, but perhaps Kingo Takiguchi, Jean Sévigny, and (citric acid) Leonidas Emmanouilidis in particular. In the mid-sixties I had to measure Pi in the presence of all the unstable organic phosphates in algal cells of which ATP was by no means the least stable. I cannot find my thesis right now, but I believe an acid somewhat weaker than HCl (or even TCA) was used, and timing was precise. Colour was observed for some 30 minutes but the data extrapolated back to the instant the acid was added. This seemed to correct for a steady background rise in apparent Pi.
So it turned out that, apparently, I started with a "good" malachite green reagent. Today I tested 4 different protocols (thanks to everyone providing them/helping me to set them up) and in all the negative controls (ATP only) I observed a dramatic breakdown of ATP over time that people described except with the malachite green reagent I initially used. With this reagent there was also spontaneous hydrolysis of ATP but by far less than with the other 3 tested reagents. After 1 hour of keeping it on the bench there was still a clear and significant difference between +protein and -protein samples while one could not differentiate anymore between sample and negative control when using any of the other reagents.
Also, does anyone know what polyvinyl alcohol exactly does when used in an ATP hydrolysis assay? I am asking because there was a remarkable difference in the Pi quantification between two reagents that contained PVA compared to two reagents that did not. I found in the literature that PVA allows the quantification of Pi without having it separated from the protein and that PVA prevents the precipitation of the protein. However, knowing more about why it does it would be helpful!
No clue but make sure that all compounds that you test do not interfere with the malachite assay. We had in the past a std curve that changed (still linear but with a different slope) with a thiophosphate compound if I recall well.
Most of the color drift is due to non-enzymatic ATP hydrolysis under acidic conditions imposed by the reagent. Another, less likely, but nevertheless formally possible contributor is some acid-stable ATPase contaminant (ATPases are abundant enzymes!).
Here are the fixes:
1) You need to use a color stabilizer (sodium citrate), which acts by raising the pH of the mixture after the addition of the reagent. Add it in 1 min after the reagent. (see references below for details)
2) Mix/measure absorbance at constant time intervals for most reproducible results. The 30-min time between the quench and measurement that you are using is good. (people already suggested this in this thread)
3) You may want to try different ratios of reaction:reagent volumes to see what works best.
Here are the references with the details. We have experimented a lot with this assay and find it very reliable, reproducible and quantitative. I hope it works for you as well.
Biswas T, Resto-Roldan E, Sawyer SK, Artsimovitch I and Tsodikov OV. (2013) A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG. Nucleic Acids Res., 41, e56.
Biswas T, Pero JM, Joseph GC, Tsodikov OV. (2009) DNA-dependent ATPase activity of bacterial XPB helicases. Biochemistry, 48, 2839-2848.
I had experienced the same problem with the Malachite Green Phosphate Assay kit. Can anyone let me know if 10-30 mM EDTA or 0.5% SDS can be used in the assay to stop the ATPase? I am thinking about quenching samples at different time points before adding MGR at roughly the same time point, then 1/10 volume of 34% Na Citrate for color stabilization after 1 minute.
Ailong,
Chelation of the magnesium ion with excess EDTA should stop the ATPase reaction.
Magdalena,
I would appreciate if you could send the detailed protocol for ATPase assay to [email protected]
Marco, what you think is residual activity, is likely one or both of the following: 1) a reagent (color) instability or 2) non-enzymatic hydrolysis of ATP in the acidic solution due to the acidity of the reagent, instead. As Magdalena noted, your protein should be inactivated by the acid. A solution for 1) is to use polyvinyl alcohol in your malachite green reagent recipe and 2) use a Na citrate challenge after the quench with the reagent.
A protocol that works well can be found here:
Biswas T, Pero JM, Joseph GC, Tsodikov OV. (2009) DNA-dependent ATPase activity of bacterial XPB helicases. Biochemistry, 48, 2839-2848.
Dear Oleg Tsodikov,
It would be very useful to know what is the best protein concentration for malachite green ATPase assay. Because today my protein got precipitated. It was 4mg/mL concentration protein(crude extract of yeast).
Dear Roopa,
The enzyme concentration is generally dependent on how enzymatically active your protein is, how your reaction is carried out (manually vs with fast mixing) and conditions (such as concentration of substrate and reaction time). For example, the ATPase reaction that you monitor should produce at least 5-10 microM Pi in a given reaction time for a robust measurement by the malachite green reagent method.
The Malachite Green reagent is a strong acid. If the protein concentration is high enough, the protein will precipitate. 4 mg/ml is definitely high enough to precipitate. An additional problem is that the ATPase activity in a crude extract is likely to come from multiple enzymes.
Note that there is no need to buy a kit for Malachite Green assay. It is easy to perform when a few details are known. I will be happy to provide a detailed protocole and protocol also if protein precipitation is really necessary. But we never had to do it ourselves. Indeed nucleotides degrades into Pi in acidic conditions (but not Pi itself that is stable in acidic condition so the STD curve will be OK for a day but not nucleotides where high PI background will occur after just 20-30 min.
Regards
Dear Oleg,
Thank you. I never mixed in between the reaction time of 3hrs. Only in the beginning I do mixing. I will have to mix I feel reading your answer.
The assay protocol I follow is Martin G Rowlands,2004.
Adam,
Thank you. I will change today to 1mg/mL and check.
Jean,
If you have the protocol for the ATPase assay please send to me [email protected].
I would like to compare.
Thank you
3 hrs reaction? You need to make sure that the reaction is linear for all that time. For NTDases and NPPS we do reaction never more than 30 min. Generally 20 min. The reaction is stable for these enzymes for 30min to 1hr so to be safe.
I will send the protocole shortly. Wishes.
Respected Magdalena,
plz forward me the detailed protocol for ATPase assay at my email id [email protected]
Can someone send me a detailed protocol for malechite green assay for free orthophosphate formed during ATPase activity.
I would like to compare the current protocol i am using with another one.
email id: [email protected]
I have used the Malachite Green/molybdate reagent described in this paper:
Itaya and Ui (1966) Clin. Chem. Acta. 14, 361-366
It is stable at room temperature in the dark for months and provides sensitivity between 0 and 20 µM Pi. Because the pH is so low, the reagent will cause ATP or other nucleotides to break down slowly, so the absorbance should be read about 5 minutes after reagent addition. Free phosphate in nucleotide stock solutions limit the concentration of nucleotide that can be used.
There are many other reports of Malachite Green/molybdate reagents in the literature.
Hi Marco, can you please send me the protocol that Madalena sent you? Has it worked for you? Thanks! e-mail: [email protected]
When I did my ATPase activity assay , I mixed malachite green reagent on ice, in the ratio of 1:1:2:2 0.572% ammonium molybdate/6N HCl:0.232% ployvinyl alcohol:0.0812% malachite green:ddH2O, but normally it should change color from brown to green for half or one hour, doesn't ? But my MGR reagent keep to be brown , only shows a little lighter after one hour. I don't know what's reason? Can anyone help me ?
Can someone kindly send me a detailed protocol for malechite green assay for ATPase activity please......... via my e-mail address:
Please, see this manuscript for a detailed malachite green protocol to measure ATPAse activity:
Biswas T, Pero JM, Joseph GC, Tsodikov OV. (2009) DNA-dependent ATPase activity of bacterial XPB helicases. Biochemistry, 48, 2839-2848.
Hi all
Anyone know the role of each component in the test? what is the role of Malachite green , Ammonium molybdate, Triton x-100 and citric acid?
Regards
From this web page: http://bitesizebio.com/7214/ask-a-chemist-how-colorimetric-assays-work/
This simple assay method is based on the complex formed between malachite green, ammonium molybdate, and free orthophosphate (aka inorganic phosphate, Pi) under acidic conditions.
(see the web page for the scheme)
Orthophosphate, liberated from a phosphorlyated substrate upon cleavage by the phosphatase, forms a complex with ammonium molybdate in a solution of sulfuric acid. The formation of the malachite green phosphomolybdate complex, measured at 620-650 nm, is accordingly directly related to the free orthophosphate concentration. Therefore, it is possible to quantify phosphorylation and phosphate release from protein phosphatase substrates.
Things to Keep in Mind…
This assay measures only inorganic free phosphate; organophosphates (lipid-bound or protein-bound phosphates) must first be hydrolyzed and neutralized prior to measurement. Knowledge of the different classes of organophosphates and their respective different free energies of hydrolysis aids in the optimization of assay conditions. Higher-energy organophosphates (acetal phosphates, phosphoanhydrides, etc ) possess acid-labile phosphate groups that can be released into solution by incubation at low pH. On the other hand, lower-energy organophosphates (phosphoesters) are stable in acidic solution and require much harsher conditions (such as thermal decomposition) to be detected by this method.
In the presence of a large excess of malachite green, the 3:1 ion associate ((MG+)3(PMo12O40 3-)) can easily form and precipitate in the acidic aqueous solution. To stabilize the 1:1 ion associate in the aqueous solution, polyvinylalcohol is added to the solution.
Another thing to take into consideration during the assay is the possibility of any redox reactions that could interfere with the assay. Molybdenum is a transition metal and so exists in many oxidation states. In the molybdate anion, it has an oxidation state of +6. Reduction of the acidified Mo(VI) solution by organic compounds, like ascorbic acid and reducing sugars (i.e. glucose), or by inorganic compounds, like SnCl2, creates the Mo(IV) species, which is blue in color.
------------------------
This answer doesn't cover Triton X-100 and citric acid. I would have to see the text of the article to comment. Unfortunately, that article by Biswas et al is not open-access, and I don't want to pay $40 to read it.
Triton X-10 is often added to assays done in polystyrene microplates to help keep proteins from sticking to the surface of the plate. It may also be used to help keep otherwise insoluble proteins in solution. Citric acid (as citrate ion) can be used as a metal ion chelator to stop metal-dependent reactions.
There are many versions of the Malachite Green/molybdate reagent in the literature and available commercially. Although they all contain molybdate and Malachite Green, obviously, they differ in other components.
Dear Adam
Thanks for your answer, the attached id the Biswas, et al 2009 paper.
best wishes
Thanks for the paper, Hadi. The procedure for the phosphate assay includes polyvinyl alcohol, which is often included to help stabilize the reagent. Sodium citrate is added just after the molybdate/Malachite Green reagent to prevent any further color development caused by acid hydrolysis of nucleotides. Citrate complexes with remaining free molybdate.
The method referenced in Biswas et al is in this paper by Lanzetta et al:
http://download.bioon.com.cn/upload/201103/21/223257b7ff75bok7s9yotu.attach.pdf
Dear
I would like to recommend you the following publication: Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB. Seetha V. Balasingham, Ephrem Debebe Zegeye, Håvard Homberset, Marie L. Rossi, Jon K. Laerdahl, Vilhelm A. Bohr, Tone Tønjum. PLoS ONE 7(5): e36960.
Thank
Marius
Can someone kindly send me a detailed protocol for malachite green assay for phosphatase activity plz zzz ..via ([email protected])
Sir I want to estimate ATPase concentration in serum, so what is the best method?
Rakesh,
HPLC or TLC are correct methods as detection of Pi by malachite green or other Pi determination assays will be a problem with serum.
Unless may be if you dialyze sufficiently the sample....
For those who would like it we can send our detailesd protocole of Malachite green assay. Jsut ask to :
If you want to quantify the amount of a specific ATPase protein in serum, rather than the total ATPase activity from any source, the best way would probably be to set up a sandwich ELISA. This would require having two antibodies to the protein with non-overlapping epitopes.
If you want to measure the ATPase enzyme activity in serum, the Malachite Green assay probably won't be useful because the acidity will precipitate a lot of serum protein. There are many other ATPase assay methods that may be better for this purpose, including a wide variety of commercial kits. Here is one example that claims to be useful for serum samples:
http://www.abcam.com/atp-assay-kit-colorimetricfluorometric-ab83355.html
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
In your protocol, I don't see an important step, adding sodium citrate to raise pH. There are a few things that are going on in the reaction after you add the malachite green reagent: 1) the enzyme activity is quenched by the very low pH (as Magdalena noted), 2) the reagent binds inorganic phosphate and 3) ATP is hydrolyzed by the acid. Because 2) is not instantaneous (it takes ~20 min to develop color at microM Pi), you need to wait. On the other hand, as Adam noted, you will need to measure at the same time after you add reagent to the reaction for each data point. The sodium citrate will help reduce nonenzymatic ATP hydrolysis. As Adam wrote, the assay is very reproducible, but you need to mix things well and keep track of timing. I posted our detailed protocol in this thread somewhere, for your reference.
Hello Magdalena Sch, can you please share the detail protocol at [email protected]. it would be a great help.
I followed the original method of Lanzetta et al. (1979 at Analytical Biochemistry), but I could not find Sterox, so that I used Tween 20. I had a problem that absorbance kept increasing for several hours after 34% sodi@um citrate. I am wondering that anybody had similar problem or had any suggestions??? Your responses will be much appreciated. Thanks.
My question is that how to determine different allergens in fish and shrimp muscle? Second is .... which instrument would be best suited to determine allergens?
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