ab105134 Alanine Transaminase Activity Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of Alanine Transaminase activity in various samples. This product is for research use only and is not intended for diagnostic use. Version: 3 Last Updated: 6 March 2014
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Table of Contents 1. Overview 3 2. Protocol Summary 4 3. Components and Storage 5 4. Assay Protocol 7 5. Data Analysis 9 6. Troubleshooting 11 2
1. Overview Alanine transaminase (ALT) is a transaminase enzyme (EC 2.6.1.2). It is also called serum glutamic pyruvic transaminase (SGPT) or alanine aminotransferase. (ALAT) is found in serum and in various bodily tissues, but is usually associated with the liver. It catalyzes the reaction: α-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 Abcam s Alanine Transaminase Activity Assay Kit, ALT catalyzes the transfer of an amino group from alanine to α-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 (λ 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 ALT with a detection limit of 10 mu per well. 3
2. Protocol Summary Standard Curve Preparation Sample Preparation A Add Reaction Mix Measure Optical Density 4
3. Components and Storage A. Kit Components Item Quantity ALT Assay Buffer 25 ml OxiRed Probe (in DMSO) 0.2 ml ALT Enzyme Mix (Lyophilized) 1 vial ALT Substrate (Lyophilized) 1 vial Pyruvate Standard (100 nmol/µl) 0.1 ml ALT positive control (Lyophilized) 1 vial *Store the kit at -20 C, protect from light. Allow ALT Assay Buffer to warm to room temperature before use. Briefly centrifuge vials before opening. Read the entire protocol before performing the assay. ALT ENZYME MIX: Reconstitute with 220 μl dh 2 O. Aliquot and store at -20 C. Use within two months. ALT SUBSTRATE: Reconstitute with 1.1 ml Assay Buffer. Aliquot and store at -20 C. Use within two months. 5
ALT POSITIVE CONTROL: Reconstitute with 100 μl dh 2 O. Aliquot and store at -20 C, use within two months. In the assay (optional), add 5-10 μl positive control and adjust the final volume to 20 μl/well with ALT Assay Buffer. B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker 6
4. Assay Protocol 1. Standard Curve Preparation: a. For the colorimetric assay: Dilute the Pyruvate Standard to 1 nmol/μl by adding 10 μl of the Standard to 990 μl of ALT 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, 2, 4, 6, 8, 10 nmol/well of the Pyruvate Standard for the colorimetric assay. b. For the 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 Pyruvate 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. 2. Sample Preparation: a. For tissue or cell samples: Tissues (50 mg) or cells (1 x 10 6 ) can be homogenized in 200 μl ice-cold ALT Assay Buffer, then centrifuged (13,000 x g, 10 min) to remove insoluble material. b. For serum samples: Serum samples can be directly diluted in the Assay Buffer. 7
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. 3. Reaction Mix: Mix enough reagents for the number of assays to be performed. For each well, prepare a total 100 μl Reaction Mix: ALT Assay Buffer 86 μl OxiRed Probe 2 μl ALT Enzyme Mix 2 μl ALT Substrate 10 μl Add 100 μl of the Sample Reaction Mix to each well containing the Samples, Standards, and Positive Controls (optional). Mix well. Note: The fluorometric assay is ~10 times more sensitive than the colorimetric assay. Use 0.4 µl of the probe per reaction to decrease the background reading & increase detection sensitivity significantly. 4. Measurement Read OD 570nm (A 1 ) at T 1 (T 1 >10min) then again (A 2 ) at T 2 after incubating the reaction at 37 C for 60 min (or longer if the ALT activity is low), protect from light. The OD of the color generated by oxidation of pyruvate is: ΔA 570nm = A 2 A 1. 8
Note: It is recommended that the user run the assay kinetically to choose A 1 and A 2 values which occur after the initial lag phase, during the linear range of color development. OD at A 2 should not exceed the highest OD in the standard curve. 5. Data Analysis Plot the pyruvate Standard Curve and use the ΔA 570nm to obtain B nmol of Pyruvate. ALT activity in the test samples can then be calculated: ALT Activity = B (T 2 T 1 ) x V = nmol/min/ml = mu/ml Where: B is the pyruvate amount from pyruvate Standard Curve (in nmol). T 1 is the time of the first reading (A 1 ) (in min). T 2 is the time of the second reading (A 2 ) (in min). V is the original sample volume added into the reaction well (in ml). One unit of ALT is defined as the amount of ALT which generates 1.0 μmol of Pyruvate per minute at 37 C. 9
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6. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet 11
Sample readings are outside linear range Concentrate/ dilute samples to be in linear range Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet 12
Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) Problem Reason Solution Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit 13
For further technical questions please do not hesitate to contact us by email (technical@abcam.com) or phone (select contact us on www.abcam.com for the phone number for your region). 14
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