Sialic Acid (NANA) Assay试剂盒(ab83375)

I am pleased to let you know that the sialic acid standards are now available separately under the product number ab170150.

To our knowledge only mammalian samples have been tested with this kit and we are not sure whether crude plant extracts would be compatible. The kit is designed to detect free sialic acid.

We have used this kit only with mammalian samples, therefore we cannot comment on how well it would work with plant samples. Since the principle is the same, this kit should work with samples from multiple sources including plants. However, you may need to optimize the protocol a bit for such samples.

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Yes, the SA will get out into the filtrate. That is the whole purpose of filtering this. The SA will get out into the filtrate and the higher MW interfering agents will be stuck at the top of the filter.

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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You could try the following protocol for gycoprotein, glycolipid or glycan samples:

Sample is dissolved in a final concentration of 2 M HOAc and heated to 80°C for 3 hours to release sialic acids. The released sialic acids are collected by ultra-filtration through a 3,000 NMWCO filter

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 very much for your inquiry about the ab83375.

This is a very good question.

I am sorry however that this kit will not be suitable for your needs. Indeed, as you mention, for measuring the bound sialic acid, the sample must be hydrolysed first to free the sialic acid. To do this, the samples are dissolved and you would not be able to see the localisation in the confocal microscopy study anymore.

Unfortunately we do not have an alternative kit or antibodies to measure sialic acid. Please do however not hesitate to contact us again, could we help searching for another productor advise you otherwise.

Thank you for contacting us. Could you please describe more fully the problems your customer is encountering? Does she observe the expected results when using the Sialic acid standard provided with the kit? In the protocol provided with the kit there is a detailed explanation of how these control experiments can be performed. Is the signal observed higher than expected, or lower? In answer to your questions: 1. So, she would like to know if there's something to make interference effect. Any pyruvate in the sample may cause interference. This is why carrying out a control experiment without adding Sialic acid converting enzyme is advised to observe the background signal due to pyruvate in the sample. 2. Let her know detail protocol of "Hydrolysis procedure" for measure bound sialic acid. The hydrolysis procedure can be carried out with a final concentration of 2M Acetic acid (HOAc). This sample should then be heated to 80°C for 3 hours to release the sialic acids. The solution should then be passed through a 3,0000 NMWCO filter. Using HCl as your customer has may not be suitable as it is a stronger acid and may break down the Sialic acid itself. 3. She would like to know the principal of detection sialic acid because has doubt about the possibility it detects something else. The kit uses an enzyme coupled reaction in which the free sialic acid in the sample is oxidised. This then results in the conversion of the Oxi-Red probe to give fluorescence (Ex/Em=535/587 nm) and absorbance (O.D.=570 nm). Unfortunately the exact enzyme reaction used for this process is proprietary but it is specific for Sialic acid. Pyruvate can however interfere and cause a background signal as described previously.   Could you confirm with the customer that the kit is being stored in the correct way? It should be protected against light (Sialic Acid Probe (Red cap) should be protected from light and moisture). Care should also be taken to keep the Sialic Acid Converting Enzyme (Purple cap), Development Mix (Green cap) and Sialic Acid Standard (Yellow cap) on ice during use.   Any additional information your customer has communicated to you would be useful in more fully understanding the problems she has been encountering. Many thanks for your assistance in this matter.

Thank your for your interest in this product. In answer to your questions: 1. Her sample is cell culture supernatant. Can it be used without any preprocessing? Cell culture supernatant should not require pre-processing in order to utilise the kit if measuring free Sialic acid. The levels which can be accurately measured by this kit is within the range of 0.1 to 10 nmol and optimisation of the experimental setup may be required in order to measure within this range. 2. This kit detects both free and binded sialic acid? She need total Sialic acid now. The kit measures free sialic acid but bound sialic acid can be measured by carrying out a hydrolysis procedure as follows: Sample is dissolved in a final concentration of 2 M HOAc and heated to 80oC for 3 hours to release sialic acids. The released sialic acids are then collected by ultra-filtration through a 3,000 NMWCO filter. The starting material for this procedure can be glycoprotein, glycolipid or glycan. 3. Converting Enzyme, one of the components, does it detect binded sialic acid? The role of the converting enzyme is to aid in ascertaining the level of pyruvate you have in the sample. Unfortunately, the exact role of the converting enzyme is proprietary. If a significant amount of pyruvate is suspected in your sample, this may create background and it would be advisable to carry out a background control. This can be performed by carrying out the analysis of a sample without the Sialic Acid Converting Enzyme. This will detect only the endogenous pyruvate, but not Sialic Acid. The pyruvate background should then be subtracted from Sialic Acid measurement.    I hope this information has been of help to you. If you require any further information please do not hesitate to ask.

Thank you for your inquiry. I can confirm that the sample types tested for this kit are cell and tissue culture supernatants, urine, plasma & serum, and other biological fluids. This does mean media, and not lysate. I f your customer is planning on using this kit on lysed cells, the senior scientist says that you can use the assay buffer in the kit to lyse the cells. However, your customer will then need to choose a specific neurominidase in order to cleave bound sialic acids off of glycoproteins and complex carbohydrates in preparation for using the kit. (Most sialic acid is bound and not free.) Your customer will need to choose the specific neurominidase themselves because different neurominidases will cleave at specific sites, depending on the targets. A site that the senior scientist gave me as an example is the following: http://www.bioresearchonline.com/product.mvc/Alpha-2-3-Neuraminidase-0002#. You can see that this particular neurominidase cleaves at the non-reducing terminal a(2-3) unbranched sialic acid residues from complex carbohydrates and glycoproteins. Please let me know if you have any other questions.