Catalase Activity Assay Kit (Colorimetric/Fluorometric) (ab83464)

Regarding the HRP  The amount of HRP once reconstituted in 200 ul is enough for 1 plate + 1 extra reaction (2 ul per reaction). This should therefore be enough for a 1 plate assay.   The 1 extra reaction amount means that if for example you want to split your experiments into 2X  separate assays using half a plate, you will need to be careful how much you use for the first assay and perhaps prepare master mix for half plate plus half a reaction.

The assay is measuring the amount of H2O2 left behind as it is used up by the catalase. The higher the catalase activity, the less H2O2 will be left at the end of the assay when it is measured.  The reason to add extra H2O2 is to ensure that the readings of the High Control (HC) and sample is within the standard curve.   For example, on the example shown on the datasheet, the sample data starts with OD 1.5 (HC) while the sample OD is 0.25. the difference in OD values is therefore almost near the upper end of the standard curve. It is to help ensure the sample values remain in the range of the assay if they contain a lot of catalase.

The High Control (HC) is a well that has the same volume of sample (2-78 uL) loaded as your sample wells. If you load wells with different volumes of sample, you will need to also load multiple HC wells with the same volume of sample. Prior to adding hydryogen peroxide to all of the wells, 10 uL of stop solution is added to each HC well and incubated for 5 minutes at 25 degrees Celsius to completely inhibit the catalase activity (step C). The HC well OD reading is used to calculate the signal changes by catalase in your samples as outlined in the Data Analysis section (page 9), serving as a baseline OD reading.

This is essentially a similar principle to subtracting a background OD reading (negative control), but since there is an inverse relationship between the OD reading and catalase activity with this assay, it is referred to as a High Control rather than a negative control.

Gracias por contactarnos.

En el kit tiene lugar la reacción de H2O2 para producir H2O y oxigeno catalizado por la enzima catalasa. El H2O2 que queda sin reaccionar reacciona con OxiRed™ generando un producto que presenta absorbancia medida a 570nm.

En el pocillo del High Control la reacción de conversión de H2O2 en agua y oxigeno se detiene antes de que tenga lugar, por eso se añade la Stop Solution. De esta forma, se puede saber la cantidad de H2O2 que había inicialmente en las muestras, sin que haya sido catalizada por la catalasa.

Al final, la absorbancia debida a la actividad de la catalasa vendrá dada por la siguiente ecuación: Abs (A)= Abs (HC) – Abs (simple).

La forma de preparar el HC es la misma que en las muestras: se añade la muestra primero (o el control positivo) y se ajusta el volumen hasta 78ul con Assay Buffer. Después se añaden 10ul de Stop Solution solo a los pocillos del HC, para inhibir la actividad de la enzima.

Espero haber aclarado la pregunta. En caso contrario no dudes en volverme a contactar.

Thank you for contacting us.

I can confirm that the fluorimetric detection method can be used with the 545/590 filter. Please note that the expected results could be of less quality as reading results with a 535/587 filter would be.

I can confirm, that for unknown samples, we suggest testing several doses of the sample to ensure the readings are within the linear range.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Thank you for contacting us. I am sorry to hear that you have had problems with our Catalase Assay Kit (ab83464)

I would like to reassure you that this kit is tested and covered by our 6 month guarantee for Tissue extracts. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

A. General Information
- Catalogue number: ab83464

- Lot number:

- Purchase order number/ order date or preferably the Abcam order number:

- How was the kit stored?


B. Description of the problem
- Are the signals you obtained with control or standard satisfactory? (Yes/No);


- Are the sample readings as expected or inconsistent?


- Are the blank readings as expected?

- Other (e.g. missing components)


C. What do you think the problem is caused by? E.g. enzyme is faulty, developer is faulty or other component that you think might be faulty.



D. Sample
- Sample Type (e.g. Cell culture supernatant, Cell lysates, Tissue lysates or Serum samples)


- Sample Species (e.g. human, mouse or rat)


- Storage (at what temperature and for how long were the samples stored?)

- Sample treatment, if any



- Sometime proteins in samples interfere with readings. Have you removed proteins?


- Sample preparation what dilution the sample was used in for the assay?


E. Protocol used:
Did you follow the protocol exactly as given on the datasheet?


F. Optimizations attempts (problem solving); please describe any deviations from the protocol.



G. Have you used the same kit successfully before?


H. Do you obtain the same results every time?


I. Additional Notes; (any additional information you would like to provide)



J. Results: I would appreciate if you could also provide any data which would help us to assess the results.



Thank you for your time and cooperation. We look forward to receiving the completed questionnaire and I hope we can find a solution soon.

Thank you very much for your interest in our products.


To our knowledge, we have do not have a Catalase Assay kit in our catalog that is tested and guaranteed for plants. Since ab83464 is working by measuring the the unconverted H2O2, which then reacts with OxiRed™ probe to produce a product, which can be measured at 570 nm (Colorimetric method) or at Ex/Em=535/587 nm (fluorometric method), this kit should be suitable also for plant catalase.

https://www.abcam.com/index.html?datasheet=83464 (or use the following: https://www.abcam.com/index.html?datasheet=83464).

This has bot been tested by us and therefore is not guaranteed.

However by participating in our AbTrial program your customer can now use our products in an untested application or species without financial risk.

Simply follow these easy steps below to apply for our AbTrial Program:

1. Reply to this email, letting us know your customer is interested in testing this product and with the customer email address and institute.

2. Our scientists will email you an inactive personal discount code for the value of the product.

3. Purchase and test the product at the regular price.

4.Your customer submits their results, including the discount code in the additional notes section of your Abreview.

5. Once the Abreview is submitted, the discount code will become active.

6. Apply the discount code on your next order for this customer to receive that value off.

Please let me know if you have any questions about this offer and I would be happy to help you and your customer further.

The Terms and Conditions of this offer can be found at: www.abcam.com/abtrial.

Thank you for your reply.


As for your samples, the dilution is extremely important and from your previous results it seems that 1:20 is best of the three you have already tested. Perhaps testing 1:25 and/or 1:30 would also be useful to check.


I hope this information is helpful and I apologize for the delay in responses. Please do not hesitate to contact me if you have any additional questions or concerns.

Thank you for your patience as I have investigated this issue further with the laboratory.


I can confirm that you performed the assay correctly the second time, only adding stop solution to the standards and HC ONCE.


It is not clear however, why the HC readings were lower than your samples. Were these freshly prepared samples? Do you have more samples and room on the plate to try again? If not, please provide an order number and I will send a replacement kit.


I look forward to your reply so that I may assist you further.