Free Glycerol Assay Kit (ab65337)

Ab65337: Este kit puede usarse a partir de extractos celulares. Para ello, comenzar con un número aproximado de ˜2X10^6 células, suspender el pellet en 500ul (o aproximadamente 4 volúmenes) de assay buffer en hielo usando un homogeneizador (10-15 pases) hasta que se haya alcanzado una lisis eficiente, lo cual puede comprobarse al microscopio.

Centrifugar la muestra y recoger el sobrenadante para utilizarse en los ensayos posteriores. Se recomienda probar distintas diluciones de muestra para asegurar que las lecturas caen en el rango linear de la curva de estándares.

Ab1333130: En este caso no hemos probado el kit con extractos celulares, no podemos sugerir un protocolo concreto de lisis, pero pueden utilizar un protocolo estándar de extracción, similar al anterior, y optimizarlo para cada caso.

No, I don’t think preparing samples once for both assays is a good idea. For the glycerol assay, you just need cell homogenates, whereas for the triglyceride assay you are actually preparing the lipid extracts from the cells and using them for further steps. You cannot use cell homogenates with the triglyceride assay and the lipid extracts for the glycerol assay. They are not interchangeable. So prepare samples for each assay as recommended and follow the protocol.

Yes, with the Free Glycerol Assay Kit, you can use HEPES for lysis, although the assay buffer provided with the kit is recommended.

With the Triglyceride Quantification Kit, we would recommend using HEPES and NP-40 since the NP-40 is important for breaking the cells.

Thank you for your inquiry and your patience with this reply. I will be helping you further as Carolyn is out of the office.
I spoke to our development scientist about this issue and they thought that the urine contains some compound which may be blocking the assay development. The fact that the glycerol and the standards are working shows that the kit is working well.
We also have not received any complaints from the use of this kit in urine so far, so I think there should not be a problem with such sample type, albeit it depends on the way the urine was processed and stored. Can you please send us some raw data and I can consult with our scientists again in order to try and figure out what could be happening?
I look forward to your reply.

Thank you for your inquiry.

The exact description of the components of this kit is proprietary information.

But I am happy to confirm the way this kit works in more detail:

The probe that is used in kit ab65337is a highly sensitive and stable probe for H₂O. It is a fluorogenic (EX/EM = 535/587 nm) and Colorometric (l = 570 nm) substrate for peroxidase.
In the presence of horseradish peroxidase (HRP), the probe reacts in a 1:1 stoichiometry with H2O2 to produce highly fluorescent resorufin.

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

Thank you for your inquiry.

Please find the answers to your questions below:

1.)This Glycerol Assay Kit provides a sensitive, easy assay to measure free glycerol concentration in various samples. In the assay, glycerol is enzymatically oxidized to generate a product which reacts with the probe to generate color (λ= 570 nm) and fluorescence (Ex/Em = 535/587 nm). The kit can detect 50 pmol-10 nmol (or ˜1-10000 µM range) of glycerol in various samples.

I attached the protocol booklet for more information

2.) Free glycerol can be measured with this kit in a wide variety of samples. If I understand you correctly, you are asking for bound glycerol in tri-ester of glycerol thatpart offats and oils?

The kit most probably does not detect these since only free glycerol can be detected. But this kit has not been specifically tested with tri-esters of glycerol.

I hope this information is helpful andwish you good luck with your research.

Thank you for contacting us.

I am happy to let you know that the Free Glycerol Assay Kitab65337does not contain any hazardous substances.

If you wish we could provide the MSDS for DMSO which would state that it is not a hazardous substance or mixture according to Regulation (EC) No. 1272/2008 ('This substance is not classified as dangerous according to Directive 67/548/EEC.')

I hope this information is helpful. Should you require the MSDS or have any further questions, please do not hesitate to contact us.

Thank you for your reply. I have contacted our Distributors Team and they will issue 1 free of charge replacement of ab65337 on your next Abcam order. I checked the lots we have in stock and this will come from a different lot but the same QC batch from the lab. We would guarantee this under the Abpromise, so do let me know if the customer has any other trouble. I hope this information helps. Please contact us with any other questions.

All of the kits: ab65337, ab65342, ab83390, ab83394 are suitable for cell and tissue culture supernatants. As well as many other biological fluids like urine, plasma and serum.

Thank you for your enquiry. It would be best to use fresh sample when possible however I know that it may take time to collect all the necessary sample before running an assay. Therefore, I would recommend storing the sample at -20 °C or colder for long term, just because we do not know if any lysis has occurred or any enzymes leaked out. Generally, in an enzyme-free sample, the glycerol should be stable for a long time (6-12 months). I hope this information will be helpful. If there is anything else that I can help you with, please do not hesitate to contact me.