D-Lactate Assay Kit (Colorimetric) (ab83429)

Nous n’avons pas de données sur la conservation/stabilité du D-lactate cependant nous recommandons de centrifuger vos échantillons d’urine à 4 ºC à 10,000 g pendant 2 minutes. Puis vous pouvez déprotéiniser vos échantillons avec notre column 10 kDa (ab93349) pour minimiser la dégradation de la lactate. Ensuite vous pouvez stocker vos échantillons à -80ºC jusqu'à utilisation. En général, vos échantillons seront stable pendant 1 mois à -80ºC.

Thank you for contacting us. Yes, D-lactate is produced in very low levels in mammals unless there are certain bacterial infections or trauma. L(+)-Lactate is the major stereoisomer of lactate formed in human intermediary metabolism and is present in blood. The lactate to pyruvate ratio reflects the redox state of the cell and describes the balance beween NAD+ and NADH, which is dependent on the interconversion of lactate and pyruvate via lactate dehydrogenase (LDH). Please let me know if you have any further questions.

Urine samples would also have NADH. Therefore, the background control step should be run.
Since NADH is one of the reaction intermediates in this assay, we want to make certain that any readings we see are due to the assay reaction of D-Lactate conversion and not the pre-existing NADH in the samples. Therefore doing the background control and subtracting its readings from the final sample readings will account for any pre-existing NADH.

Besides completely depressing the plunger on the pipette tip, the lab suggested also the following:

In addition, the customer can try to point the pipette tip towards the side walls of the wells instead of the bottom. That way the bubbles would most likely stick to the walls and burst before they reach the bottom.

Also, if the customer has access to a plate centrifuge, giving the plate a swift spin also helps in getting rid of some bubbles in the bottom of the well.

Thank you for taking the time to complete our questionnaire and providing this further information. I am sorry to hear you have had difficulty obtaining satisfactory results from our D-Lactate Assay Kit (Colorimetric) (ab83429) and our Pyruvate Assay Kit (ab65342).

The details you have kindly provided will provide us with vital information for our monitoring of product quality. I appreciate the time you have spent to do so.

As the standard curve is working well, in this case I would be concerned about the sample preparation. I can confirm I would expect the deproteinisation with PCA will degraded the proteins and the acidic environment will inactivate all added enzymes as already suggested. If it is necessary for this experiment to use PCA we would recommend to neutralise the PCA before proceeding (for example potassium hydroxide). Can you confirm if this has been done?

The 10kDa spin column separates out the analytes from any protein sample without degradation of proteins. For that reason we would highly recommend to use Spin columns for the sample preparation if possible at all.

I hope this information is helpful for you. Please do not hesitate to contact me if you have further problems or questions.

Thank you for your enquiry regarding ab83429.

As long as the band width of detection on the instrument says ± 50, you can use 500 nm instead of 450 nm. Otherwise it will compromise the efficiency of the assay.

I hope this helps and if I can assist further, please do not hesitate to contact me.

Please let the Dr B. know the following:

You can directly use different doses of serum/plasma with this assay. Just make up the volume in the wells to 50 µl with the assay buffer.

All readings have to be made at 450 nm. Please do not use 570 nm for it. We will update the datasheet accordingly.

This kit is not designed for HTS, so unless you yourself optimize the protocol, you won’t be able to use it with a fully automatic random Access analyzer.

Yes, you can run different doses for the sample. Just add different volumes of the sample to different wells for analysis.

Hope this information has been helpful for you.

Please let me know if you have any other questions.

Thank you for contacting us.

I am not familiar with the contents of MRSB media, but in general cell culture supernatants will be compatible with these kits. If the media contains serum, we recommend deproteinizing the samples using our 10 kDa cutoff spin columns (ab93349) to prevent degradation of the lactate. Phenol red is generally not a problem because the samples will be substantially diluted, but you can always add some diluted media to your blank wells to control for any additional background.

I hope this helps, please let me know if you need any additional information or assistance.

Thank you for your inquiry.

1.) I can confirm that urine should be centrifuged for 30 minutes at 1500g. Then the samples should be stored at -80C and / orde- proteinised with ab93349 (10kD spin column).

https://www.abcam.com/10kD-Spin-Column-ab93349.html (or use the following: https://www.abcam.com/10kD-Spin-Column-ab93349.html).

2.) We do not have any data showing the Chlorhexidine might affect the results.

I hope this information is helpful and wish you good luck with your experiments.

Thank you for your inquiry.

I am not personally familiar with the Roche Cobas C501, however this is a colorimetric assay, so as long as your plate reader measures at 450 nM, it should be fine.

The protocol with all the volume infois linked below:

https://www.abcam.com/ps/products/83/ab83429/documents/ab83429%20D%20Lactate%20Assay%20Kit%20Colorimetric%20(Website).pdf

I hope this information helps. Please contact uswith anyother questions.