Thioredoxin Reductase Assay Kit (Colorimetric) (ab83463)

Thank you for contacting us and your interest in our products.

I can confirm this product is sourced externally and the molar concentration of the TrxR inhibitor is proprietary information. Regrettably we are not allowed to chare this information on this occasion.


I am sorry that I can not be of more help in this case. If you have any further questions, please do not hesitate to ask.







proprietary information, which unfortunately I cannot share with you.

Ab83463 can be used to measure TrxR in plasma. It does not need any special preparation. It can be used directly or diluted depending on the TrxR content.

Several volumes must be tested in a pilot assay to ensure readings are within the linear standard curve range. Total protein content of plasma can be measured as an internal normalization method.

I wanted to let you know that I have been informed by the lab that there is EDTA in this kit.

Thank you for contacting us.

The standard in this kit is from rat.

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

Thank you for contacting us.

I can confirm, that regrettably we can not provide any data using serum samples with this kit. However, every sample type and application stated for a product on our datasheets is covered by our six month guarantee. If you experience any problems we would be pleased to offer technical support. In case the ab83463 is not working as stated on our datasheet we will be pleased to offer a free of charge replacement or a credit note as compensation.

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I apologise for the confusion with our troubleshooting tips. The troubleshooting guide is a general guide provided with all our kits. I can confirm that for the Thioredoxin (TrxR) Reductase Assay Kit (ab83463) it is not recommended to use a >10 kDa Spin column, out of the reasons you already mentioned. Due to your valuable feedback we updated our product booklet and deleted this point in the troubleshooting section.


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

I have had some feedback from the lab to share with you which may help to improve the results seen so far.


The assay buffer needs to be allowed to come to room temperature before use. This may have significantly reduced the reactivity observed with the assay. You are however correct in thinking the NADPH, TrxR inhibitor andTrxR positive control should be kept on ice during the assay.


The kit is also not compatible with using fluorometric measurement (I assume this is what you have been doing as you are using black sides/clear bottomed plates). Using this kit you can measure the samples only colorimetrically. The lab has suggestedmeasuring the absorbance at412 nm may be better for your experiment. This should be performed using clear plates. This may significantly change your results.


There should be no problem in flash freezing the samples following preparation and storing them at -80°C till needed.


I hope these suggestions will help in producing better results than those observed so far. However, if you continue to have problems please do not hesitate to let me know.

Thank you for providing that information.

Could I ask you confirm a few additional details?

1. How was the kit stored for the month in which it was not used?

2. In particular, how was the NADPH kept, both before reconstitution and after? How long in total do you think it was kept on ice when carrying out the assay?

3. Could you also label both what the graph represents and what each of the wells had in it?In particular thestandard used (0, 10, 20, 30, 40 and 50 nmol standard) as well as with the inhibitor added? This will allow me to see if this is the data we would expect to see with the standard experiments.

4. I think I've located your order, was it delivered to the University of Sussex around the 27th of October?

Many thanks again for your continued cooperation.

Thank you for contacting us and reporting the problems experienced with the Thioredoxin (TrxR) Reductase Assay Kit (ab83463). From having spoken to you over the phone, there does not immediately appear to be an obvious solution to what you have been observing. In order to understand the problem further, and hopefully to be able to pinpoint what may be going wrong, could you please answer the following questions:

ORDER

1. Could you please provide me with the order number or the approximate delivery date of the kit.

SAMPLE

1.What cells are you using to perform this experiment?
2.How many cells are used for every experiment?
3.Are they cultured as a suspension or as adherent cell culture?
4. How were the cell lysed? Was protease inhibitor used?

PROCEDURE

1.What volume of sample was added to each well for analysis?
2.How was the reaction mix prepared and how much was added?
3. Was the assay buffer allowed to equilibrate to room temperature before use?
4.Whatmethod was used for measurement?Fluorescent, Luminescence or Colorimeter? And at what wavelength?
5. Which kind of plate was used?
6. Did you perform a reaction kinetics with a sample to find the linear range? At what time points was t1 and t2 taken?

STORAGE

1. How was each of the constituents stored following reconstitution?

a. DTNB
b. NADPH
c. TNB Standard
d. TrxR Assay Buffer
e. TrxR Inhibitor
f. TrxR Positive Control

DATA

Could you please share with me the data you have obtained using the kit, specifically the standard curve generated, the kinetic curve generated with your samples, and the readings generated with your samples. This will be very helpful in understanding what you have been observing.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Thank you for contacting us. The Thioredoxin (TrxR) Reductase Assay Kit ab83463 contains a TrxR specific inhibitor that is utilized to determine TrxR specific activity. Unfortunately, we have not tested the kit or the inhibitor with insect cells. Theoretically based on the conservation of this enzyme between species, it should also work with insect cells although is is optimised for human samples. Hence in this case the customer will have to determine if this inhibitor would also work for TrxR from insect cells. In case there were any problems using this kit with insect cells, we are happy to offer technical support. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for contacting us.  The Thioredoxin (TrxR) Reductase Assay Kit ab83463 contains a TrxR specific inhibitor that is utilized to determine TrxR specific activity. Unfortunately, we have not tested the kit or the inhibitor with insect cells. Theoretically based on the conservation of this enzyme between species, it should also work with insect cells although is is optimised for human samples. Hence in this case the customer will have to determine if this inhibitor would also work for TrxR from insect cells. In case there were any problems using this kit with insect cells, we are happy to offer technical support. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.