实验概要

Alanine  Transaminase (ALT) is a transaminase (EC 2.6.1.2) also called serum  glutamic pyruvic transaminase (SGPT). Alanine Transaminase is found in  serum and in various body tissues, but is usually associated with the  liver. It catalyzes the reaction: alpha-ketoglutarate alanine =  glutamate pyruvate. It is commonly measured clinically as a part of a  diagnostic liver function test to determine liver health.  Diagnostically, it is almost always measured in units/liter (U/L). In  ab105134, Alanine Transaminase catalyzes the transfer of an amino group  from alanine to alpha-ketoglutarate, the products of this reversible  transamination reaction being pyruvate and glutamate. The pyruvate is  detected in a reaction that concomitantly converts a nearly colorless  probe to both color (lambda max = 570 nm) and fluorescence (Ex/Em =  535/587 nm). The kit provides a rapid, simple, sensitive, and reliable  test suitable for high throughput activity assay of Alanine Transaminase  with a detection limit of 10 mU per well.

主要试剂

ALT Positive Control  [Detail：Lyophilised]

ALT Substrate [Detail：Lyophilised]

Pyruvate Standard (100 nmol/µl)

ALT Assay Buffer

ALT Enzyme Mix  [Detail：Lyophilised]

OxiRed™ [Detail：in DMSO solution]

实验步骤

1.      Reagent preparation:

1) Alanine Transaminase Enzyme Mix: Reconstitute with 220 µl dH2O. Aliquot and store at -20oC. Use within two months.

2) Alanine Transaminase Substrate:   Reconstitute with 1.1 ml Assay Buffer. Aliquot and store at -20oC. Use within two months.

3) Alanine  Transaminase Positive Control: Reconstitute with 100 µl dH2O. Aliquot  and store at -20oC, use within two months. In the assay (optional), add  5-10 µl positive control and adjust the final volume to 20 µl/well with  Alanine Transaminase Assay Buffer.

2.      Standard Curve Preparation:

1) Colorimetric assay:

Dilute  the Pyruvate Standard to 1 nmol/µl by adding 10 µl of the Standard to  990 µl of Alanine Transaminase Assay Buffer, mix well. Add 0, 2, 4, 6,  8, 10 µl into a series of standards wells. Adjust volume to 20 µl/well  with Alanine Transaminase Assay Buffer to generate 0, 2, 4, 6, 8, 10  nmol/well of the Pyruvate Standard for the colorimetric assay.

2) Fluorometric assay:

Dilute  the Pyruvate Standard to 1 nmol/µl as for the colorimetric assay. Then  dilute the standard another 10-fold to 0.1 nmol/µl by taking 10 µl into  90 µl of Pyruvate Assay Buffer. Mix well. Add 0, 2, 4, 6, 8, 10 µl into a  series of standards wells. Adjust volume to 20 µl/well with ALT Assay  Buffer to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well of the Pyruvate  Standard for the fluorometric assay.

3) Sample Preparations:

Tissues  (50 mg) or cells (1×106) can be homogenized in ~ 200 µl ice-cold  Alanine Transaminase Assay Buffer, then centrifuged (13,000 g, 10 min.)  to remove insoluble material. Serum samples can be directly diluted in  the Assay Buffer. Prepare test samples of up to 20 µl/well with Assay  Buffer in a 96-well plate. We suggest testing several doses of your  sample to make sure the readings are within the standard curve range.

4) Reaction Mix:

Mix enough reagents for the number of assays to be performed. For each well, prepare a total 100 µl Reaction Mix:

Alanine Transaminase Assay Buffer     86 µl

OxiRed Probe                       2 µl

Alanine Transaminase Enzyme Mix    2 µl

Alanine Transaminase Substrate       10 µl

Add  100 µl of the Sample Reaction Mix to each well containing the Samples,  Standards, and Positive Controls (optional). Mix well.

5) Measurement: 

Read  OD 570 nm (A1) at T1 (T1 >10min) then again (A2) at T2 after  incubating the reaction at 37 °C for 60 min (or longer if the Alanine  Transaminase activity is low), protect from light. The OD of the color  generated by oxidation of pyruvate is DeltaA570 nm = A2 - A1. It is  recommended that the user run the assay kinetically to choose A1 and A2  values which occur after the initial lag phase, during the linear range  of color development. OD at A2 should not exceed the highest OD in the  standard curve.

6) Calculation:

Plot  the pyruvate Standard Curve and use the DeltaA570 nm to obtain B nmol  of pyruvate (amount of pyruvate generated between T1 and T2 in the  reaction wells). Alanine Transaminase activity in the test samples can  then be calculated:

Alanine Transaminase Activity = B/(T1-T2)xV = nmol/min/ml = mU/ml

Where: B is the pyruvate amount from pyruvate Standard Curve (in nmol).

T1 is the time of the first reading (A1) (in min).

T2 is the time of the second reading (A2) (in min).

V is the original sample volume added into the reaction well (in ml).

One  unit of Alanine Transaminase is defined as the amount of Alanine  Transaminase which generates 1.0 µmol of pyruvate per minute at 37 °C.