Alanine Transaminase Activity Assay Kit (Colorimetric/Fluorometric) (ab105134)

Overview

  • Product name
    Alanine Transaminase Activity Assay Kit (Colorimetric/Fluorometric)
  • Detection method
    Colorimetric/Fluorometric
  • Sample type
    Cell culture supernatant, Urine, Serum, Plasma, Other biological fluids, Tissue Extracts, Cell culture media
  • Assay type
    Enzyme activity (quantitative)
  • Sensitivity
    > 10 mU/well
  • Assay time
    1h 20m
  • Product overview

    Alanine Transaminase Activity Assay Kit (Colorimetric/Fluorometric) ab105134 is a rapid and simple assay used to quantify alanine transaminase (ALT) activity in mammalian samples.


    In the ALT assay protocol, ALT transfers an amino group from alanine to α-ketoglutarate; producing pyruvate and glutamate. The pyruvate is detected in a reaction that converts a nearly colorless probe to a form that is colored (ODmax = 570 nm) and fluorescent (Ex/Em = 535/587 nm).


    The kit has a detection limit of 10 mU per well.


    ALT assay protocol summary:
    - add samples and standards to wells
    - add reaction mix and incubate for 10 min at 37ºC
    - analyze every 2-3 min for 60 min with microplate reader in kinetic mode at 37ºC

  • Notes

    Alanine transaminase is also called alanine aminotransferase or serum glutamic pyruvic transaminase (ALT, ALAT, SGPT).

  • Platform
    Microplate reader

Properties

Images

  • Serum aspartate transaminase (AST) and alanine transaminase (ALT) levels were measured at various time periods post Con A injection. Mean values ± SD are shown (n = 4). ∗P < 0.05 and ∗∗P < 0.01.
  • Liver samples from high fat diet (HFD) and standard carbohydrate diet (CHD) BALB/c and C57BL6/J mice were homogenised in an ALT assay buffer for the determination of ALT activity using ab105134. A separate batch of liver extracts was prepared and incubated in a buffer containing NP40 (5%) and supernatants containing the triglycerides were separated. Triglycerides concentration was determined on the supernatant fraction using ab65336. ALT activity and triglycerides concentration were determined by measuring OD at 570nm.

  • Colorimetric standard curve: mean of duplicates (+/- SD) with background reads subtracted.
  • Fluorometric standard curve: mean of duplicates (+/- SD) with background reads subtracted.

  • Alanine transaminase measured in mouse tissue lysates showing quantity (mU) per mg of tested sample.

    Protein concentration for samples varied from 4 mg/mL to 13 mg/mL. Samples were diluted 9-27 fold and measured colorimetrically.

  • Pyruvate measured colorimetrically in cell lysate after 20 min and 40 min incubation time showing quantity (nmol) per 1 mln of tested cells.

  • Measurement of alanine transaminase in HepG2 cells (10 μg) and liver lysate (15 μg).

  • Pyruvate measured fluorometrically in cell lysate after 20 min and 40 min incubation time showing quantity (nmol) per 1 mln of tested cells.

  • Pyruvate measured in biological fluids after 20 min and 40 min incubation time showing quantity (nmol) per ml of tested sample.

  • Pyruvate measured in mouse tissue lysates after 20 min and 40 min incubation time showing quantity (nmol) per mg of tested sample.

Protocols

References

This product has been referenced in:
See all 15 Publications for this product

Customer reviews and Q&As

1-10 of 18 Abreviews or Q&A

Question
Answer

The precipitate can be re-solubilized by gentle heating with occasional swirling. Placing in hot water bath of 50 - 60 degree for 10-15 min would be more than enough. Avoid vigorous shaking as there may be foam formation.

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Abreviews
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Verified customer

Submitted Jan 24 2018

Question
Answer

J’ai eu une réponse du laboratoire qui m’a confirmé que ce kit peut être utilisé sur des échantillons humain.

Malheureusement le laboratoire n’a pas de conversion entre l’activité et la concentration d’ALT. mU est équivalent à nmol/min d’ALT et le poids moléculaire d’ALT est 54.47.

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Answer



Ich möchte hiermit auch bestätigen, dass beide Kits für Zellkulturüberstand getestet und auch garantiert sind.

Sie haben Recht, dass eine Probenaufbereitung nicht gesondert erwähnt wird.

Zellkulturüberstand (frei von Zellen) kann direkt eingesetzt werden. Wir empfehlen einen unverdünnte Probe und dann einige verschiedene Verdünnungen zu testen, damit man sicherstellt, dass die Werte in den Bereich der Standardkurve fallen.

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Answer

I am sorry for the delay in getting back to you. I now have some further information to share with you.

Contrary to what I mentioned in my previous email, the L-Alanine Assay Kit (ab83394) is unlikely to be of use in the experiments you are performing. This is because an alanine transaminase is not used in the conversion of the substrate. I am sorry for any confusion caused in this. The Alanine Transaminase Activity Assay Kit (ab105134) should however be applicable if you amend the protocol.

As discussed, you may need extra ALT Positive Control as the kit is not designed for the experiments you are hoping to perform but to measure ALT activity within biological samples. I would be able to add this component to the catalogue for you if you would like. This would cost £100 for 1 vial (as supplied with the whole kit).

I hope this has been of help. Please do let me know if I can be of any further assistance.

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Answer

Thank you for contacting us yesterday in regards to the Alanine Transaminase Activity Assay Kit (ab105134). I have now had a chance to have a look at this product to see if it would be suitable to use in your experiment to assess the transaminase inhibition properties of your compounds.

In short, the kit should be compatible with this but would need to be modified in order to get the results that you are looking for. The kits is intended to be used to measure the inherent alanine transaminase activity of biological samples. For example, measuring the activity from liver tissue samples. This is performed by using the provided substrates for the enzyme to work on and measuring the pyruvate generated. The enzyme comes from the biological sample tested.

You would therefore need to introduce the alanine transaminase enzyme into your samples to measure the inhibition caused by your compounds. This enzyme is provided as the ALT positive control. What you would need to do is set up the reaction with this included in each of your wells and add your inhibitors. You could then compare the inhibition by observing the difference of the ALT activity in the presence and absence of inhibitor.

As the kit is not really designed for this purpose it may require some optimisation and the quantity of ALT provided is not very high as it is only intended to be used as a single positive control. You may want to consider looking at the L-Alanine Assay Kit (ab83394) instead. This kit allows for the detection of alanine but works on the same principle as ab105134. It uses ALT to convert alanine to pyruvate which is then specifically detected.

Again, this kit is not specifically desigend for the experiment you are hoping to perform and therefore the protocol would need to be modified. In this kit, the alanine standard is provided but may again be limiting in the experiment you are hoping to perform.

What I am going to do is see if either the ALT positive control for ab105134 or the alanine standard from ab83394 may be available to purchase independently of the kits. This may help you as you would be able to run more tests with each kit. Once I have this information I will get back to you.

I hope this information has been of help. If you have any further questions in the meantime, please do not hesitate to let me know.

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Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this kit is tested and covered by our 6 month guarantee for. In the event that a product is not functioning as cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

As discussed on the telephone, I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you are also able to provide any data and graphs which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.



A. General Information
- Catalogue number:


- Lot number:

- Purchase order number/ order date or preferably the Abcam order number:

- How was the kit stored?


B. Description of the problem
- Are the signals you obtained with control or standard satisfactory? (Yes/No);


- Are the sample readings as expected or inconsistent?


- Are the blank readings as expected?

- Other (e.g. missing components)


C. What do you think the problem is caused by? E.g. enzyme is faulty, developer is faulty or other component that you think might be faulty.



D. Sample
- Sample Type (e.g. Cell culture supernatant, Cell lysates, Tissue lysates or Serum samples)


- Sample Species (e.g. human, mouse or rat)


- Storage (at what temperature and for how long were the samples stored?)

- Sample treatment, if any



- Sometime proteins in samples interfere with readings. Have you removed proteins?


- Sample preparation


E. Protocol used:
Did you follow the protocol exactly as given on the datasheet?


F. Optimization attempts (problem solving); please describe any deviations from the protocol.



G. Have you used the same kit successfully before?


H. Do you obtain the same results every time?


I. Additional Notes; (any additional information you would like to provide)



J. Results:

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Answer

Thank you for contacting us.

You are correct, the first readin should be done at RT after 10 minutes. After this you can incubate at 37 degrees away from light before taking the second reading. We have not tested this product using RO water and cannot give you any data supporting its use.

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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Question
Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxxxxx

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.

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Question
Answer

Thank you for your inquiry.

I heard back from the lab that whole blood cannot be used with this assay kit as the color will disrupt the reading. You will have to get plasma/serum to use it.

I hope this information helps. Please contact us with any other questions.

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1-10 of 18 Abreviews or Q&A

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