GST Assay Kit (Colorimetric) (ab65326)

Ich möchte hiermit auch bestätigen, dass beide Kits für Zellkulturüberstand getestet und auch garantiert sind.

Sie haben Recht, dass eine Probenaufbereitung nicht gesondert erwähnt wird.

Zellkulturüberstand (frei von Zellen) kann direkt eingesetzt werden. Wir empfehlen einen unverdünnte Probe und dann einige verschiedene Verdünnungen zu testen, damit man sicherstellt, dass die Werte in den Bereich der Standardkurve fallen.

Thank you for your enquiry and your interest in our products.

This kit is not specific for any GST isoforms so it detects the total GST enzymatic activity in the samples (using CDNB substrate).

There are some other GST substrates available but most of these compounds have overlapping specificity to the different forms. Your customer may be able to find some published articles on this topics using PubMed::

http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1033.2002.03141.x/full

If you need any further assistance in the future, please do not hesitate to contact me.

Thank you for your enquiries which have been forwarded to me as my colleague Karin is currently away from the office.

I am sorry to hear you have had some difficulties with this kit and for the inconvenience this has caused. As agreed with Karin, I have issued a free of charge replacement of 2 X GST and 2 X assay buffer. The order number is 1211009.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that these free of charge replacement vial are also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Thank you very much for your reply and reporting the moisture you have noticed with the GSH.

The lab are going to test some stock of this compound to investigate this solubility problem you have been experiencing further. I will get back to you with this information as soon as I can. I have also forwarded your observation of the moisture in the vial to see if this is likely to have contributed to the problem, or if it is likely to detrimentally affect the assay.

I am sorry for the delay. I will get back to you as soon as possible.

Thank you for your reply.

I am glad to hear your subsequent runs with the kit have worked out well. I am still waiting on further information from the lab to advise you further in regards to the precipitate found in the GSH component and will of course pass this on to you as soon as I can. I am sorry for the delay.

Thank you for your reply.

I have asked my colleagues in the US to look into if we can offer any discount on the order. One we can certainly offer free of charge as I offered to you previously. They should be in contact soon to arrange for the order to be put through.

If I can be of any further help please do let me know.

Thank you for your reply.

I cannot currently find a reference to the "nmol" but it seems this may have been an error. The protocol booklet should read as in the attached document (with the GST activity units in μmol/min/ml).

I am sorry for all the confusion caused.

Sorry for the delay in my reply.

The units calculated for the GST activity should be in μmol/min/ml. The μmol unit comes from the extinction coefficient. I could not find any reference in out protocol to nmol. Where have you found this? I'd like to makesure there is not a mistake in the protocol.

I look forward to receiving your reply.

Sorry for the delay in getting back to you.

I now have feedback in regards to the calculations you are trying to perform:

The source of the kit has confirmed that thesecond of your calculations accordingis the closest to being correct. Here is theircalculation:

4.9 x 0.105 divided by 0.0096 x 1000 x 0.2893 x 0.003 =0.5145 by 0.0083318 = 61.75 umol/min/ml.

Thedilution factor was taken as 1 because you areadding pure undiluted 3 ul of the protein into the wells. Its addition with the buffer and the mix is accounted for in the 0.105 ml calculation.

Now each ml ofyour samplewill have a total of 6 mg of protein (6ug/ul = 6 mg/ml).

Therefore the reading is 61.75 umol/min/6mg. Which is equivalent to 10.29 umol/min/mg.

Additionally, wethink that the2.3 to 3.5 “Vmax” changeshould be significant if you put it into calculations. I would make sure to do enough repeat readings to have confidence in the figures and that it is not simply down to sample variations.

Also, if you could provide me with your order number (or purchase order number) I can arrange to reimburse you for the value of a kit. Would you prefer for this to be in the form of a credit note (to be used against future Abcam orders) or as a refund?

Hope this information has been helpful for you. If I can be of any further assistance, please do not hesitate to let me know.

Thank you for getting back to me and sorry for the delay in my reply.

Hopefully I can now sort out dome of the confusing information. There has been an error in the equation provided to calculate GST. Unfortunately, the extinction coefficient was entered incorrectly and should be 0.0096 umol-1cm-1, not 0.0023 as it was changed to in the most recent booklet. The 0.2893 was added to include the pathlength of the solution. The units are in µmol/min/ml. This information will be updated in our protocol as soon as possible. I am very sorry for the inconvenience this confusion has caused. I hope the equation now makes sense to you. Asan apologyfor the confusion we have caused youI would like to offer you a free GST Activity Assay kit in compensation. Please let me know if you would like for me to arrange this.

In answer to the change in GST assay buffer in the kits. The buffers are different, this change was made to improve the kit. However, the results from the previous version of the kit are going to be comparable with this new version. Please do not interchange the components from one version to the other and you should be fine. I try to get the information regarding the composition of the GST buffer for you but I think it will probably be proprietary information but I will let you know either way.

I have also arranged for the components you have asked for to be added to our catalogue. These are available under the following catalogue numbers:

CDNM component ab139170

GSH component ab139176

In answer to your question of how you can convert the activity to relate to the amount of protein I can answer the following:

If you are starting with 1 mg of protein in 100 ul the concentration would be 0.1 mg/ml or 0.1 ug/ul, Then if theyou aretaking 0.006 ml which is 6 ul, and you are therefore starting with with 0.6 ug of the protein. The final volumes you dilute it to then does not matter, as long as this is the working solution. You should therefore not need to multiply any of the dilution values.

Does this make sense?

I look forward to receiving your reply.