Questions and answers concerning the applications of our ATP determination kit

1. How do I have to calibrate the assay for exact ATP determination with a standard curve?
2. We do not have a luminescent reader - can I use the ATP determination kits nevertheless?
3. What are the advantages of the ATP determination Kit PRO?
4. Regarding the samples preparation (extraction method), which type of buffer can I use?
5. How much cell lysate do I have to add to the ATP reagent mix for ATP determination in bacteria or cell cultures?
6. What standard protocols for ATP determination in bacteria or cell cultures do you suggest?
7. Which components in my cell lysis buffer interfere with the luciferase reaction in your ATP determination kits?
8. Are there other extraction methods of ATP in bacterial cultures?
9. Are there any additional advices when working with tissue samples?
10. Can I use the ATP determination kits for enzymatic reactions and for binding studies of ATP to recombinant proteins?
11. Do you provide a literature list of publications for the use of the luciferase reaction for ATP determination?
12. Peer-reviewed publications citing use of this product
13. Can I contact somebody for special applications of the ATP determination kits not mentioned in the FAQ list?

Q1. How do I have to calibrate the assay for exact ATP determination with a standard curve?
Aug 29, 2018

In order to accurately determine ATP concentrations, it is necessary to generate a standard curve for a series of defined ATP concentrations. For the determination of unknown ATP concentrations you should always use the same experimental conditions (temperature, incubation times, assay volume, luminometer adjustments, etc.) for yielding reproducible results. Highest reproducibility of data is achieved, when luminescence is directly read after a fixed incubation time of 10 minutes.
The ATP Determination Kit PRO is optimised for a total amount of 0.1 to 500 pmol ATP per assay with nearly constant luminescence signals over a period of up to two hours. Higher ATP concentrations should be diluted to obtain optimal results.
Preparation of 100 mM ATP stock solution for standard curves:
We recommend the use of Adenosine-5-triphosphat Disodium salt (min. 99%). For volumes of 50-100 ml stock solution, an appropriate quantity of the salt is dissolved in 100 mM 3-(N-Morpholino)-Propansulfonacid (Mops) pH 7.0.
To prevent ATP hydrolysis, the acid solution should quickly be neutralised to pH 7.0 using concentrated NaOH.
The exact ATP concentration of the stock solution is determined using a photometer at 260 nM (e=15000 (mol/l)-1 cm-1).
Aliquots of ATP stocks can be stored at -18 degrees for six months (avoid multiple freeze thaw cycles).

Q2. We do not have a luminescent reader - can I use the ATP determination kits nevertheless?
Dec 05, 2006

As an alternative to a luminometer a scintillation counter can also be used to measure luciferase activity. Make a significant dilution of the sample (in 1x reaction buffer supplemented with 1mg/ml BSA) in a clear or translucent vial so that the sample completely covers the bottom of the vial. The sample can also be placed in a microcentrifuge tube in the vial. Do not add scintillant! For a linear relationship between luciferase concentration and counts per minute (cpm), the coincidence circuit on the scintillation counter should be turned off. If it can not be turned off, you have to calculate the square root of measured cpm minus background cpm using a water or buffer blank ([sample-background]1/2). The scintillation counter must be used in manual mode and should be read individually for 1-5 minutes each.

Q3. What are the advantages of the ATP determination Kit PRO?
Aug 29, 2018

The bioluminescent assay ATP determination Kit PRO combines the previous sensitive and the time-stable assay ATP determination kit. The assay enables a sensitive detection of very small amounts of ATP (down to 0.1 pmol) with an optimized luminescent signal stability for up to two hours.

Q4. Regarding the samples preparation (extraction method), which type of buffer can I use?
Dec 05, 2006

There are many examples in the literature for the application of luminescent detection of ATP in cell cultures of many different species. According to the relatively high stability of luciferase, many buffer components can be used which do not influence the functionality of the assay, although the complex composition in cell extracts can have an effect on the sensitivity of the assay.

Q5. How much cell lysate do I have to add to the ATP reagent mix for ATP determination in bacteria or cell cultures?
Dec 05, 2006

Our ATP determination kit have been tested with bacterial cells from E. coli and with eucaryotic COS7, HEK and Jurkat cells. For bacterial and eucaryotic cells with estimated ATP levels of about 1-2 mM a number of 1e3 to 1e6 living cells per assay/well is recommended to be in the optimal range. The assay volume to be applied depends on the cell density. Equal volumes of cell lysate and final ATP reagent should be mixed to yield a final assay volume of 100 µl (50 µl lysate + 50 µl ATP reagent), but it is also possible to use final assay volumes of 20 µl (10+10) or 400 µl (200+200) and any volume in between. For an accurate ATP determination an ATP standard curve should be analysed as well (works best in the same buffer that you use in the final experiments).

Q6. What standard protocols for ATP determination in bacteria or cell cultures do you suggest?
Dec 05, 2006

Cell lysis can be performed by several freeze-thaw cycles, however, that procedure does not suppress ATPase activity completely. Therefore, the luminescent signal will decrease within one hour even though the time stable assay has been used. More reproducible results can be achieved when samples are spun down carefully, resolved in component D buffer of our kits used as lysis buffer and heated for 5 min at 95 degrees Celsius which results in both complete lysis of cells and blockage of ATPases.
For this purpose you can order component D separately from the ATP determination kit in volumes of 30 ml (http://www.proteinkinase.biz/page.php?modul=GoShopping&op=show&category=70&article=188-reaction buffer, 30 ml) or 100 ml (http://www.proteinkinase.biz/page.php?modul=GoShopping&op=show&category=70&article=189-reaction buffer,100 ml).

Q7. Which components in my cell lysis buffer interfere with the luciferase reaction in your ATP determination kits?
Dec 05, 2006

Care has to be taken when measuring ATP concentrations in cell extracts. After cell lysis endogenous ATP will be degraded rapidly without inhibition of ATPases and the luminescent signal will decrease significantly within one hour. Freeze-thaw techniques, french-press or other extraction methods often use cell lysis buffers containing different detergents and metal chelating agents. Although luciferase needs Mg2+, 1-2 mM EDTA or EGTA can be used in the cell lysis buffer. Many detergents even enhance activity of luciferase and the assay mix also contains non-ionic detergents. Try to avoid anionic detergents like SDS because in higher concentrations this type of detergents inhibit the luciferase enzyme (see "The Effect of Detergents on Firefly Luciferase Reactions", Simpson WJ, Hammond JRM, J Biolum. And Chemolum. 1991, 6: 97-106).
Cell lysis buffers may contain DTAB (0.05 - 2%), BDDABr, sulfobetaine 3-10, SDS*, deoxycholate* (*concentrations only up to 0.05%) and NaF (1-20mM) as ATPase inhibitors, cell-lysing agents like TCA, DMSA, CTAB or ethanol and enzyme stabilising agents like THESIT (1-5%), BSA or gelantin.

Q8. Are there other extraction methods of ATP in bacterial cultures?
Dec 18, 2008

Extractions were performed in small glass tubes using 0.9 mL of various extractants. The tubes were equilibrated for 2 min at ambient or elevated temperatures using a temperature-controlled water bath. Thereafter, 0.1 mL bacterial culture was rapidly pipetted directly into the extractant solution to achieve immediate mixing. The following extractants were used: Tris–EDTA buffer containing 0.1 mol/L Tris(hydroxymethyl)aminomethane and 2 mmol/L EDTA and adjusted to pH7.75 with acetic acid; dodecyl trimethyl ammoniumbromide (DTAB) (1%, 2% or 4%) dissolved in Tris–EDTA buffer; TCA (10%) dissolved in distilled water; NaOH (1 mol/L) dissolved in distilled water; and DMSO, used undiluted. In the finally adopted method, 0.9 mL 2% DTAB in Tris–EDTA buffer was equilibrated for 2 min in a boiling water bath and 0.1 mL sample was added. After heating for 1 min, extracts were allowed to cool down to ambient temperature in the air. Extracts were stored in the refrigerator or on ice if kept for a few hours or in the freezer if kept for longer periods.
TCA, DMSO and 100°C Tris–EDTA buffer without DTAB gave around 50% of the ATP yield as compared to 100°C Tris–EDTA with DTAB. NaOH at 100°C gave a high yield but would be hazardous to work with. Furthermore, even after neutralization the resulting high salt concentration in the extract would be inhibitory in the firefly luciferase reaction. Thus it was decided to optimize the 100°C Tris–EDTA buffer with DTAB as the most promising method. Various levels of DTAB (0%, 1%, 2% and 4%) were compared, performing the extraction in a water bath temperature controlled slightly below 100°C. The optimum DTAB concentration was 2% with essentially similar yields at 1% and 4%.
The extraction with 100°C Tris–EDTA buffer containing DTAB not only gives the best ATP yield but also has a number of additional advantages:
(a) heating at 100°C is an efficient way of killing bacteria. The addition of DTAB should make the method even more safe
(b) the presence of EDTA in the Tris–EDTA buffer is known to improve inactivation of ATP-degrading enzymes and to improve extract stability
(c) The extraction ‘principles’ do not interfere with the assay. Heat is dissipated and DTAB cen be neutralized by addition of 10% a-cyclodextrin in the final ATP detection mix.
If necessary dilute this cell lysates to ATP concentrations which are most sensitive in the luminescent assay (e.g. 0.01-10µM ATP / assay PRO) and start luciferase reaction by addition of an equal volume of the final reagent assay mix (see product data sheet for details)

Q9. Are there any additional advices when working with tissue samples?
Dec 18, 2008

Our bioluminescent assay should also work with tissue sampels; however, it is essential to maintain ATP levels constant during sample preparation. Therefore addition of ATPase inhibitors (such as detergents or NaF) or pH shifts are strongly recommended for this procedure. Please check the recipe of the lysis buffer that you usually use for preparation of your tissue samples, if any of the components in the buffer will influence the detection via the luciferase reaction (see Question 7!)

Q10. Can I use the ATP determination kits for enzymatic reactions and for binding studies of ATP to recombinant proteins?
Dec 18, 2008

Our ATP determination kits measure free ATP concentrations by firefly luciferase reaction. The resulting luminescence signal is proportional to free ATP in the reaction mix. You can easily determine ATP concentrations after enzymatic reactions when the reaction is stopped prior to the luciferase reaction to prevent further consumption of ATP during luminescence measuring.
For ATP binding proteins which do not convert ATP into ADP or AMP our assays can estimate the ratio of unbound to bound ATP if the ATP is bound tightly to the recombinant protein. Weak interactions with high ATP exchange rates can not be measured. For your recombinant protein you should try to measure serial dilutions of recombinant protein with a constant ATP concentration in the assay. With higher concentrations of recombinant protein the luminescence signal should drop fast. If you measure constant luminescence even with high concentrations of recombinant protein, the ATP is not bound tight enough to determine concentration differences between free ATP in solution and protein bound ATP.
Try to use our ATP Determination kit PRO with 10 µM ATP and apropriate concentrations of your recombinant protein, use a blank with 10 µM ATP without recombinant protein and compare the results with a standard curve of 1 nM up to 10 µM of ATP to determine how much ATP is bound to your recombinant protein.

Q11. Do you provide a literature list of publications for the use of the luciferase reaction for ATP determination?
Dec 18, 2008

Some Publications to Luciferase / ATP Determination Kits:
reviews:
Clinical and biochemical applications of luciferases and luciferins, Kricka LJ., Anal Biochem. 1988 Nov 15; 175 (1): 14-21
firefly luciferases for microbiological tests (susceptibility testing, detection of bacteriuria, activated sludge analysis, food testing, immunoassays, DNA probe assay)
Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme, Stanley PE et al., Anal Biochem. 1969 Jun;29(3):381-92
Reliable luminescent detection of subpicomole amounts of ATP using a scintillation counter, Phillippy BQ, Biotechniques 1994 Apr;16(4):596-8
specific applications:
Cell viability:
Comparison of MTT and ATP-based assays for the measurement of viable cell number, Petty RD et al., J Biolumin Chemilumin. 1995 Jan-Feb; 10(1):29-34
effect of chemotheraputic drugs on tumor cell lines, microplates with low cell numbers (1500 cells/well)
The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity; Crouch SP et al., J Immunol Methods 1993 Mar 15;160(1):81-8
linear relationship between cultured cell number of MNC, MOLT-4, HL-60, TF-1, NFS-60, L-929 cells and ATP bioluminescence signal, strong correlation to uptake of tritiated thymidine as a measure of cell proliferation, ATP method can detect cytokine dependent cell proliferation (effects of GM-CSF, G-CSF, TNF)
Bacteriology:
Kinetics of bactericidal activity of antibiotics measured by luciferin-luciferase assay, de Rautlin de la Roy et al., J Biolumin Chemilumin 1991 Jul-Sep; 6(3):193-201
bacterial metabolic activity indicated by ATP production; free, extracellular or total ATP measured independently for studying bacteriostatic/bactericidal activity of antibodies or bacteriallysis (Echerichia coli ATCC 25922, Staphylococcus aureus 25923)
Hygiene Monitoring:
An enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase for the simultaneous determination of ATP and AMP (RNA), Sakakibara T et al, Anal Biochem. 1999 Mar 1;268(1):94-101
constant luminescence after stable phase is reached, ATP / AMP measurable ranges from 4*10-13 � 4*10-17 g/assay, 1*10-8 � 1*10-12 g/assay RNA by estimating the amount of AMP resulting from the degradation of RNA with nuclease P1, food samples were subjected to measurement of the amount of ATP+AMP+RNA as index for hygiene monetoring (sensitivity improved by 20 million compared to relying only to amount of ATP)
Enzymology / Kinetic:
Bioluminomertric method for real-time detection of ATPase activity, Karamohamed S et al., Biotechniques. 2001 Aug;31(2):420-5
ATPase activity of apyrase; continuous monitoring of ATP hydrolysis activity; effects of inhibitors
Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity, Karamohamed S. et al, Biotechniques 1999 Apr;26(4):728-34
Km determination for ADP and differnt nucleotides (dATP alpha S, dTT alpha S, dGTP, dTTP, dCTP, GTP) with 0.05 mU NDP kinase (real-time ATP generation is measured)
ATP in blood samples:
ATP: The Red Blood Cell Link to NO and Local Control of the Pulmonary Circulation, Sprague RS, Ellsworth ML, Stephenson AH and Lonigro AJ 1996 Am. J. Physiol. 271:H2717-H2722.
Increases in Flow Rate Stimulate Adenosine Triphosphate Release From Red Blood Cells in Isolated Rabbit Lungs, Sprague RS, Ellsworth ML, Stephenson AH and Lonigro AJ 1998. Exp Clin Cardiol. 3:73-77.

Q12. Peer-reviewed publications citing use of this product
Aug 15, 2011

Ozalp VC, Pedersen TR, Nielsen LJ, Olsen LF.(2010) "Time-resolved measurements of intracellular ATP in the yeast Saccharomyces cerevisiae using a new type of nanobiosensor."
J Biol Chem.;285(48):37579-88.
Jorgensen AL, Juul-Madsen HR, Stagsted J. (2010) "Colostrum and bioactive, colostral peptides differentially modulate the innate immune response of intestinal epithelial cells."
J Pept Sci. 16(1):21-30.
Merlin J, Evans BA, Csikasz RI, Bengtsson T, Summers RJ, Hutchinson DS. (2010) "The M3-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK-AMPK-dependent mechanism."Cell Signal.;22(7):1104-13.
Krijnen PA, Sipkens JA, Molling JW, Rauwerda JA, Stehouwer CD, Muller A, Paulus WJ, van Nieuw Amerongen GP, Hack CE, Verhoeven AJ, van Hinsbergh VW, Niessen HW.(2010) "Inhibition of Rho-ROCK signaling induces apoptotic and non-apoptotic PS exposure in cardiomyocytes via inhibition of flippase." J Mol Cell Cardiol. 49(5):781-90.
Ghanbari-Niaki A, Soltani R, Shemshaki A, Kraemer RR.(2010) "Effects of acute ethionine injection on plasma ghrelin and obestatin levels in trained male rats." Metabolism. 59(7):982-7. Epub 2009 Dec 31.
Thies E, Mandelkow EM. (2007) " Missorting of tau in neurons causes degeneration of synapses that can be rescued by the kinase MARK2/Par-1." J Neurosci. 27(11):2896-907.
Krijnen PA, Cillessen SA, Manoe R, Muller A, Visser CA, Meijer CJ, Musters RJ, Hack CE, Aarden LA, Niessen HW.(2005) "Clusterin: a protective mediator for ischemic cardiomyocytes?" Am J Physiol Heart Circ Physiol.289(5):H2193-202.
Paul K, Erhardt M, Hirano T, Blair DF, Hughes KT. (2008) "Energy source of flagellar type III secretion." Nature ;451(7177):489-92.
Krzeminski P, Suplat D, Czajkowski R, Pomorski P, Baranska J. (2007)"Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells." FEBS J. 274(8):1970-82
Sipkens JA, Krijnen PA, Meischl C, Cillessen SA, Smulders YM, Smith DE, Giroth CP, Spreeuwenberg MD, Musters RJ, Muller A, Jakobs C, Roos D, Stehouwer CD, Rauwerda JA, van Hinsbergh VW, Niessen HW. (2007) "Homocysteine affects cardiomyocyte viability: concentration-dependent effects on reversible flip-flop, apoptosis and necrosis."
Apoptosis. 12(8):1407-18.
Hutchinson DS, Bengtsson T.(2006)"AMP-activated protein kinase activation by adrenoceptors in L6 skeletal muscle cells: mediation by alpha1-adrenoceptors causing glucose uptake."
Diabetes.55(3):682-90.

Q13. Can I contact somebody for special applications of the ATP determination kits not mentioned in the FAQ list?
Dec 18, 2008

If you have further questions concerning specific lysis buffers or other applications, please contact drewianka@biaffin.de