Ascorbic Acid Assay Kit (Biological Samples) (ab65656)

Unfortunately, this kit will measure the level of Vitamin C, Ascorbic Acid, in biological samples, but not the protein you are after. Ascorbate peroxidases (or APX1) are enzymes that detoxify peroxides such as hydrogen peroxide using ascorbate as a substrate.

Unfortunately, we do not have any kits against this target available in our catalog.
I would like to recommend checking the Biocompare website which has an excellent antibody search facility that includes many suppliers. The links are:
www.biocompare.com
http://www.biocompare.com/ProductCategories/2045/Antibodies.html

I am sorry for the delay in getting back to you. I now have further input from the lab.
From the results you have shared it does seem like there is higher background in your samples than would be expected. This can often be improved by optimizing the time of the final incubation step. The protocol describes keeping this step to no more than 2.5 minutes. We would recommend that this is adhered to. Under these experimental conditions, ascorbate reacts almost instantaneously with the reagent while other antioxidants react much more slowly, so the longer the wait time the higher the background will be. It may even produce better results if you shorten this step further. The source of this kit has suggested trying this straight after having completed the mixin step (20 seconds).
If you are worried about the appropriate mixing I would suggest you could use a multi-channel pipette if you have acess to this.
I hope this information has been useful for you. Please let me know if you have any other questions.

Thank you for contacting us yesterday in regards to the Ascorbic Acid Assay Kit (ab65656). I was sorry to hear you have been having some difficulties in achieving consistent results with your samples.
As discussed over the phone, I would like to take a closer look at the data that you have been collecting in order to see if there is anything we can suggest to improve the results. To this end, if you could send the data that you have collected that would be very helpful. You mentioned that for your repeat runs you were only using 3 points for the standard, if you could also send the run where you performed the complete standard curve that would be helpful.
Could you also give a brief description of the experiments carried out. Can I also ask, following the addition of the reaction mix to the wells, how long after did you take the reading?
In answer to your question in regards to the ascorbate concentration equation, I can confirm that the V refers to your sample volume (therefore if using undiluted serum this would be 100 uL in your case, or 0.1 mL as it needs to be in mL).
I look forward to receiving your reply.

Thank you for contacting us.

The lab confirmed thata 0.1% SDS based lysis buffer can be used for cell lysis, and would be compatible with the kit.

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 contacting us. Because we carry over 80,000 products, it isn't feasible for us to keep small sample sizes of our products and kits.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

We would guarantee this kit to work to detect ascorbic acid in biological samples ranging from 0.2 to 20 nmol of ascorbic acid. If you purchase this kit and use it as described, we would be happy to replace or refund it within 6 months of purchase.

I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

Thank you for your email. Each kit contains sufficient reagents for 100 tests, therefore it is possible to test ~40 samples in duplicate with each kit (6 data points in the standard curve). Therefore you will require 3 kits for 120 samples. I hope this information helps, please do not hesitate to contact me should you have further questions.

Thank you for your patience. Yes, I can confirm that in the kit, FeCl3 is present in sufficient amount that EDTA (used at the concentration as anti-coagulant in plasma/serum samples) will not interfere with the assay. One would need to use a lot of EDTA to affect the assay. 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 your inquiry. I just heard back from the testing laboratory that these kits cannot distinguish ascorbic acid isoforms. I am sorry that this information could not be more helpful, but please let us know if we can be of any other help.

Thank you for contacting us. I have hear back from the ab with the following information to your questions: 1. Yes, you can use plasma samples – but for ab65656 we recommend removing the proteins (e.g. by using spin filter (10 kDa cutoff) (ab93349) or the Deproteinizing Sample Preparation Kit (ab93299)). 2. The proteins can be removed after thawing your plasma samples. 3. EDTA in your plasma samples is probably OK, but I am still waiting for a definite answer from the lab. I will let you know when I have the information. 4. The kit ab65656 is the best to use with your samples. 5. Yes, the power of this assay kit ab65656 is that it determines ONLY ascorbate by eliminating (subtracting) contributions for any other anti-oxidants. To do this need a background sample for EACH test sample. That means you would need three background samples for the 3 samples per patient. I hope this information is so far helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for contacting us. We do not have data from tissue samples other than serum but we assume the kit will be suitable for these samples. You will need to determine how much sample to add to keep the optical densities within the linear range of the standard curve OD. Samples prepared with BHT and DTPA have not been tested but we have no reason to believe that these will interfere with the assay, nor will thawing from -80C. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.