Every day we find new applications for the technology that is the utilization of firefly reaction

What is the shelf life of the products?

It is 2 years from manufacturing date of the oldest kit component, if you follow the storage recommendations.

Do I have to keep the kits in the freezer?

The ATP kits can be kept in the fridge until expiry date. If you prefer you can store the kits in the freezer for several years. The Luciferase Assay Kits should be kept in the freezer.

Does freezing and thawing decrease activity of the reagents?

Before reconstitution freezing and thawing does not affect the reagents. Reconstituted reagents containing luciferin can be frozen at -80°C but not at -20°C. Repeated freezing and thawing should be avoided. All other reagents and diluents including ATP standards can be frozen and thawed.

Do I have to order through your distributor or can I order directly from BioThema?

We recommend you to order via the distributor in your own country, if possible, as indicated in the products section. However, based on our long experience in luciferase assays BioThema is happy to provide detailed technical support directly to end-users.

Do your kits work in tropical conditions, Africa?

Yes, as long as they are stored and used according to our recommendations. The thermostable recombinant luciferase in our ATP kits is very stable. However, luciferin is rapidly degraded at temperatures above 25°C. At 37°C our ATP standard is degraded by 0.26% per day. Transportation, which normally is done at ambient temperature, should therefore be done with cooling to countries where the goods may be at elevated temperatures for several days.

What customs restrictions apply for your products?

So far we have not met any customs restrictions. However, with distant countries we prefer that the receiving party handles the transport and customs declaration.

What is the normal method of transportation from BioThema to the customer?

Most shipments are sent by TNT or by DPD and the cost of shipping of one kit is 15-50 EUR depending on country. We always try to keep freight cost to a minimum. Products are typically delivered 1-3 days after ordering.

Do your kits work in cold conditions, Greenland?

Yes, our reagents have actually been used in the Antarctic. Even if the assays work best around 25°C, it is possible to perform assays as long as the reagents are in liquid form. Correct results will be obtained due to the use of internal ATP standards.

Do you have CE marking?

At the moment only one of our products requires CE marking. This product is used for screening bacteria in urine, and so far we have only launched this product in Spain.

Can I use your kits for any ATP linked enzyme assay.

Any reaction that results in formation or degradation of ATP can be monitored using ATP reagents. ATP Kit SL is specifically designed to continuously monitor ATP being enzymatically formed or degraded. The ATP Reagent SL is added to the solution in which ATP is formed or degraded.

The ATP concentration is monitored by measuring the light intensity, which at any point in time is proportional to the ATP concentration. This is achieved by a luciferase activity degrading < 0.5% of the ATP per min. This assay principle has been used to measure several enzymes and metabolites as well as more complicated reactions as oxidative phosphorylation and photophosphorylation. Similarly release of ATP from cells can be continuously monitored, e.g. platelet aggregation.

I want to perform protein kinase assays. Can you provide a kit for this purpose?

We have a dedicated kit for protein kinase assays, 111-021 Kinase RR Kit.

Why do I need an internal ATP standard?

The signal from a luminometer in general is displayed as rlu (relative light units). The magnitude of 1 rlu varies between different luminometers even from the same manufacturer and often even for the same instrument model. Different ATP reagents give different light emission from the same amount of ATP. The activity of an individual ATP reagent goes down with time. Different samples contain different levels of inhibitors of the light emission. The light emission varies with the temperature. Consequently ATP can not be measured in rlu.

All these problems are solved by calibrating each individual assay by measuring the light emission before and after adding a known amount of ATP (the internal ATP standard method). The light emission from the ATP sample is affected in the same way as the light emission from the ATP standard. The ratio of these two light emission values multiplied by the amount of ATP in the ATP standard gives the amount of ATP in the sample expressed in moles rather than rlu. In this way you don’t need to prepare a standard curve for any other reason than to check the linearity of the instrument (the linearity of the light emission with ATP goes from the detection limit up to 1 µmol/L ATP). Only one ATP standard concentration is needed and should in your final assay mixture be at least 10x higher than the highest ATP concentration in your final assay mixture. The volume of the ATP standard added should be 10 µL (the lowest volume that can be added with high accuracy with most dispensers). A higher volume will dilute the assay mixture and will give less accurate results.

Do your kits work with all bacteria?

Those ATP kits that contain Extractant B/S work with all bacteria, since this strong extractant not only opens up the cell walls but also rapidly inactivates ATP degrading enzymes. The extraction is completed within seconds with all bacteria except mycobacteria. With mycobacteria it is necessary to perform the extraction on a boiling water bath for 60 seconds.

How many bacteria can you detect in water samples?

Without filtration we can detect 5 cells in a 50 µL water sample. With filtration we can detect 10 cfu/mL.

Is it possible to detect bacteria in blood, serum or cerebrospinal fluid?

Blood is difficult, since the ATP from all the blood cells has to be degraded. Serum may be possible, but we are not aware of anyone doing this. The same goes for cerebrospinal fluid.

I want to measure ATP in mammalian cell cultures. Which kit should I use?

BioThema manufactures two kits for measurements of ATP in mammalian cell cultures: 1. 155-050 Cellular ATP Kit HTS 2. 188-441 Cell Viability Kit SL Both kits have the same ATP Reagent SL normally resulting in a stable light emission and a similar detection limit (generally a single mammalian cell). The difference is the extractant used for lysing the cells to release ATP making it accessible for the luciferase reaction. Cellular ATP Kit HTS contains Lysing Diluent, which lyses the cells, but does not inactivate ATPases. Lysing Diluent therefore also contains an inhibitor of ATPases.

With high numbers of some cells, e.g. nerve cells, the inhibition is not complete and ATP will be degraded resulting in a slow decay of the light emission. If you can measure the light immediately after adding ATP Reagent SL, i.e. if you have reagent dispensers on your luminometer, this does not cause any problems.

The main advantage of Cellular ATP Kit HTS is that there is only one reagent addition. With Cell Viability Kit SL the extractant is added in the first step and the ATP Reagent SL in the second step. The extractant completely inactivates all ATPases in the sample and a stable light will be obtained. If you are uncertain which kit to choose, you can order sample kits containing only one reagent bottle of each type of reagent. Different culture media inhibit the luciferase reagent to various extents. If you have dispensers, you should use one of them to add internal ATP standard with every sample. If you don’t have dispensers, you should add external ATP standard to fresh culture medium and use the light emission from this measurement to calculate the ATP in moles in your samples. The standardisation procedures are described in Instructions for use included with the kit.

I would like to measure the extracellular ATP content in a cell culture, and need to do these measurements in the same culture for at least 72 hours and cannot change the medium at any time. So I need the luciferase to be quite stable for at least 72 hours in the medium.

Although our ATP reagents are based on a thermostable luciferase, it is not possible to add the reagent to the culture medium doing online (continuous) measurements. The reason is mainly that luciferin is not that stable. You should take aliquots of the medium for example every hour. If you mix these aliquots with an equal volume of Extractant B/S, the ATP degrading enzymes in the medium will be inactivated and you can perform the assay immediately or at a later more convenient time. All BioThema ATP kits contain a liquid-stable ATP standard. When used as an internal ATP standard you will get results expressed as pmoles and don't have to worry about a standard curve. The best product for this application is 266-311 ATP Biomass Kit HS.

Do you have any products/applications for HTS?

Most of our kits can be used for HTS provided that the customer has appropriate instrumentation. The 155-050 Cellular ATP Kit HTS is specifically intended for HTS measuring cell viability/cytotoxicity. In addition our new 111-021 Kinase RR Assay has been optimised for HTS of protein kinase inhibitors.

We are trying to achieve the lowest ATP detection limits possible, so we should be using an optimized flash reaction, correct? What should be in the buffer solution?

Flash reagents have higher luciferase activities resulting in a more intense light emission. They also consume their own background coming from ATP contamination on cuvettes and pipette tips. Therefore you can achieve a lower detection limit with flash reagents like our ATP Reagent HS (HS=High Sensitivity) and ATP Reagent SS (SS=Super Sensitivity). When selecting between the two one must also consider the ATP contamination in other reagents. In general there is little to be gained by using ATP Reagent SS, which is also more expensive.

The major concern with the buffer is not what to have in it - rather it is what not to have in it, namely ATP. Since BioThema started to produce all reagents in clean-room facilities using very special cleaning procedures for the facilities and very special operating instructions for the personnel, we have achieved much lower blanks. Obviously all chemicals in the reagents have to be extremely pure. When assays are performed no human hands should ever be in the vicinity of the reagents. Wearing disposable gloves without powder and mouth protection will take you to a long way on the route to reduce ATP contamination. Besides that we can only suggest to use the best available reagents, i.e. those available from BioThema. In this way you will take advantage of our expertise.

What technical help can I get?

We can give advice from the stage when you start to think of a project to when you write it up. We can suggest e.g. which parameters to measure and with which reagents and with which instrument. We can help you when calculating results from raw data or end results. If you want we can even help you to write a scientific report or at least parts of e.g. Materials and Methods. However, in order to help you in the best possible way we need a clear and detailed description of the problem and what resources you have available. We have long experience in helping researchers all over the world via email and we would be happy to help you in the same way. We don’t charge for this service.

Can I measure apoptosis with your kits?

We have kits for ATP/ADP/AMP determination. One of the first steps in apoptosis is that the mitochondrial ATP production is decreased. This leads to a slowly decreasing ratio between ATP and ADP in the cell. Necrosis either leads to a rapid decrease of this ratio or if the cell is killed with strong enzyme inactivating reagents that the ratio remains high.

Why do I need powder free gloves?

There is a lot of ATP on human skin. It is therefore good practice, particularly when measuring low levels of ATP, to use disposable gloves and to change these frequently. The powder is generally derived from natural products and contains a lot of ATP. This is why one should use powder free gloves.

I find it difficult to choose which kit is the best one for my application. What should I do?

Click on Kit Selection Guide, which will help you find the correct product for your application. If you are still uncertain, please send an e-mail to support@biothema.com.