实验试剂

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.