Alkaline Phosphatase Assay Kit (Fluorometric) (ab83371)

This kit is actually very sensitive, with a detection sensitivity ˜1 µU, which is more sensitive than colorimetric assays.

Since your standards worked, I do not suspect that there is a problem with the kit or your handling of the kit. The only possibility left is the sample type.

There is one slight change I can recommend. For the adherent cells, trypsinize them, take the cell pellet, wash once with PBS and then homogenize efficiently with the assay buffer using a dounce homogenizer. Do not add the assay buffer directly onto the cells and scrape them.

There is also a potential of some interfering agent in these cells, which may not have acted upon in the other kit due to a different principle.

Thank you for your enquiry regarding ab83371.

This is the buffer we recommend in our protocol to homogenize the cells/tissues, so it is used in lieu of the lysis buffer. It will not interfere with the BCA quantification.

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

For Acid Phosphatase Assay Kit ab83370, the pH of the buffer prevents alkaline phosphatase from working (ALP can only function in an alkaline pH whereas AP works in an acidic pH) so you only measure AP activity.
Acid phosphatase (AP) can dephosphorylate MUP(4-Methylumbelliferyl phosphate disodium salt) to yield 4-methylumbelliferone, a fluorescent product, with Ex/Em at 364/448 nm (pH 10.3). The reaction can be stopped by 0.3M Glycine buffer.
ab83371 : Alkaline phosphatase(ALP) can dephosphorylate MUP(4-Methylumbelliferyl phosphate disodium salt) to yield 4-methylumbelliferone, a fluorescent product, with Ex/Em at 364/448 nm (pH 10.3). The reaction occurs in the presence of glycine. So having glycine as a component of the buffer, we eliminate AP activity and only measure ALP activity in ab83371.

Hello and thank you for contacting us. The lyophilzed ALP enzyme should be stored at -20. Once you reconstitute the enzyme you should not freeze the solution. Please let me know if you have any further questions.

Thank you for your reply which has been forwarded to me as my colleague Anja is currently away from the office.

I am sorry to confirm that because we carry over 90,000 products, it is regrettably not feasible for us to stock small sample sizes of our products.

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.

I am sorry it is also not currently possible to reserve batches. However, if the customer is satisfied with one or two kits and would then like to make a bulk purchase of 5 kits or more, we would be pleased to offer a bulk discount. In this case, please do not hesitate to contact us again when the customer is ready, and we can provide a quote for you.

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

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

This kit (ab83371: Alkaline Phosphatase Assay Kit (Fluorometric)) has only been characterized on human sample; other species have not been tested yet. However, this is an enzyme-based kit so as long as your mouse myoblast cell line expresses ALP ab83371 should work. Though, optimization may be necessary since we have no data on murine system.

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

Thank you for your inquiry.

I am happy to confirm that we do have three different lots for ab83369 at this moment in time. Unfortunately, we only have one lot in stock at the moment for ab83371.

Please inform your customer that our products are for research purposes only and not validated for diagnostics.

I hope this information is helpful and wish you and your customer a good day.

Regarding the alkaline phosphatase assay that gave unusually high readings, could you please tell us how you diluted the substrate before use in the standards and samples? The protocol instructions are fairly straightforward, but we just want to be sure that you understood them, to ensure that the replacement works for you.

The lyophilized MUP substrate should be reconstituted in 1.2 ml of the provided buffer for a stock solution. This solution is then diluted 1:10 with the same buffer for the assay.

Please let me know if have any trouble with the replacement assay kit.

Thank you for confirming these details.

I have issued a free of charge replacement.

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

Please note that this free of charge replacement vial is 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 for bringing this to our attention. To help us understand the problem, I would like to ask a few questions.

You mentioned that that only the zero standard was within the limits of the plate reader's range. This was the well that did not receive any of the MUP substrate, is that correct?

Are you confident that the plate reader is set up correctly? In other words, are you or other people using the reader routinely?