Pyruvate Assay Kit (ab65342)

The laboratory responded that EDTA can interfere with the enzymatic reaction in this assay unless it can be diluted out to bare minimal.

I have spoken with the lab regarding the use of this kit in yeast and bacteria. They agree that while we have optimized this assay protocol for samples of mammalian origin, theoretically this assay should work with samples from multiple origins including bacteria and yeast. Since we have not done it ourselves, I do not know if the assay protocol is the most optimized for such samples, so you may have to do some optimization to get the most efficient results.

You can use plasma samples with this assay. Optimal dilutions have to be determined using a pilot experiment with different volumes.

Uric acid in urine is going to strongly interfere with the assay so besides doing the normal standard curve, the lab recommends to also do standard curve in the presence of X volume of diluted urine (may be 4-15 ul depends). Due to the presence of uric acid in the urine, the slope of standard curve with the urine will be different. Subtract the 0 standard from 0 Standard with urine (∆OD = 0 Standard with urine – 0 Standard). Apply this ∆OD to slope of standard curve with the urine to get pyruvate conc. in urine sample

The lab has advised me that you can keep the buffer at 37°C for some time to see if it helps. The 37°C for a short time should not adversely affect the assay; in fact many clients do keep the buffer bottle at this temp water bath while thawing it out. If you are unable to get these flakes back into solution please let us know.

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 contacting us.

Start with ˜2X106 cells, suspend the cell pellet 500 μl (or ˜4 volumes) of the assay buffer on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope. Spin down the sample and collect the supernatant. Appropriate dilutions of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve.

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

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Thank you for your email.

We recommend using this kit with 2x106 cells per 500ul of assay buffer. This cell number however is for guidelines only; the number should be optimized as it varies based on cell line type.

One way to prepare cell lysates is by adding 4 times the assay buffer to cell culture media as recommended on the datasheet. Alternatively cells can be centrifuged and then assay buffer could be added - please use the above given cell number and volume to scale down the assay buffer volume as per your requirement.

I hope this information will be helpful. Should you have any other question please do not hesitate to contact me.