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1. BioVision Alanine Aminotransferase ALT or SGPT Activity Colorimetric Fluorometric Assay Kit Catalog K752 100 100 assays Store kit at 20 C Introduction Alanine aminotransferase ALT is a transaminase EC 2 6 1 2 also called serum glutamic pyruvic transaminase SGPT or alanine transaminase ALT is found in serum and in various body tissues but is usually associated with the liver It catalyzes the reaction a 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 BioVision s ALT Assay Kit ALT catalyzes the transfer of an amino group from alanine to a 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 Amax 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 Kit Contents Es a K752 100 1 K752 100 2A K752 100 3 K752 100 4 K752 100 5 K752 100 6 ALT Assay Buffer OxiRed in DMSO at ALT Enzyme Mix lyophilized i Green ALT Substrate lyophilized Orange Pyruvate Standard 100 nmol ul Yellow ALT Positive Control lyophilized i Blue Storag2. e and Handling 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 Reagent preparation ALT Enzyme Mix Reconstitute with 220 ul dH2O 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 ALT Positive Control Reconstitute with 100 ul dH20 Aliquot and store at 20 C use within two months In the assay optional add 5 10 ul positive control and adjust the final volume to 20 ul well with ALT Assay Buffer ALT Assay Protocol Standard Curve Preparation Colorimetric assay Dilute the Pyruvate Standard to 1 nmol ul by adding 10 ul of the Standard to 990 ul of ALT Assay Buffer mix well Add 0 2 4 6 8 10 ul into a series of standards wells Adjust volume to 20 ul well with ALT Assay Buffer to generate 0 2 4 6 8 10 nmol well of the Pyruvate Standard for the colorimetric assay Fluorometric assay Dilute the Pyruvate Standard to 1 nmol ul as for the colorimetric assay Then dilute the standard another 10 fold to 0 1 nmol ul by taking 10 ul into 90 ul of ALT Assay Buffer Mix well Add 0 2 4 6 8 10 ul into a series of standards wells Adjust volume to 20 ul 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 assa
3. fter the initial lag phase during the linear range of color development OD at Az should not exceed the highest OD in the standard curve Calculation Plot the pyruvate Standard Curve and use the AA570 nm to obtain B nmol of pyruvate amount of pyruvate generated between T and T in the reaction wells ALT activity in the test samples can then be calculated B nmol min ml mU ml T2 T1 x V ALT Activity Where B is the pyruvate amount from pyruvate Standard Curve in nmol T is the time of the first reading A1 in min T2 is the time of the second reading Az 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 umol of pyruvate per minute at 37 C a g b 1 5 Masao 5 8000 4 c 14 D R m 6000 4 005 4000 O 0 0 1048x 0 0382 ae y 9078 4x 181 74 T 1 T T T 1 o 5 10 oO 0 25 05 0 75 1 Pyruvate nmoles Pyruvate nmol c 1 7 d 25 Positive control gt 20 4 3 a da 4 E 0 5 2 5104 o SE m Positive Ctrl 2 gt O Background 0 T 1 0 HepG2 Liver Lysate Time min Figure Pyruvate Standard Curve a Colorimetric b Fluorometric Measurement of alanine aminotransferase activity in Positive Control c and HepG2 Cells 10 ug and Liver Lysate 15 ug d Assays were performed fo
4. llowing the kit protocol FOR RESEARCH USE ONLY Not to be used on humans Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 2 eo onns GENERAL TROUBLESHOOTING GUIDE Problems Cause Solution o Assay not working e Use of ice cold assay buffer e Assay buffer must be at room temperature e Omission of a step in the protocol e Refer and follow the data sheet precisely e Plate read at incorrect wavelength e Check the wavelength in the data sheet and the filter settings of the instrument e Fluorescence Black plates clear bottoms Luminescence White plates Colorimeters e Use of a different 96 well plate Clear plates Samples with erratic readings e Use of an incompatible sample type e Refer data sheet for details about incompatible samples e Samples prepared in a different buffer e Use the assay buffer provided in the kit or refer data sheet for instructions e Use Dounce homogenizer increase the number of strokes observe for lysis under microscope e Samples used after multiple free thaw cycles e Aliquot and freeze samples if needed to use multiple times e Cell tissue samples were not completely homogenized e Presence of interfering substance in the sample e Troubleshoot if needed e Use of old or inappropriately stored samples e Use fresh samples or store at correct temperatures until use Lower Higher readings in Samples Bind tandade e Improperly thawed components e Thaw all comp
5. onents completely and mix gently before use e Use of expired kit or improperly stored reagents e Always check the expiry date and store the components appropriately e Allowing the reagents to sit for extended times on ice e Always thaw and prepare fresh reaction mix before use e Incorrect incubation times or temperatures e Refer datasheet amp verify correct incubation times and temperatures e Incorrect volumes used e Use calibrated pipettes and aliquot correctly Readings do not follow a linear pattern for Standard curve e Use of partially thawed components e Thaw and resuspend all components before preparing the reaction mix e Pipetting errors in the standard e Avoid pipetting small volumes e Pipetting errors in the reaction mix e Prepare a master reaction mix whenever possible e Air bubbles formed in well e Pipette gently against the wall of the tubes Standard stock is at an incorrect concentration e Always refer the dilutions in the data sheet e Calculation errors e Recheck calculations after referring the data sheet e Substituting reagents from older kits lots e Use fresh components from the same kit Unanticipated results e Measured at incorrect wavelength e Check the equipment and the filter setting e Samples contain interfering substances e Troubleshoot if it interferes with the kit e Use of incompatible sample type e Refer data sheet to check if sample is compatible with the kit or optimization is needed e Sample readings abo
6. ve below the linear range e Concentrate Dilute sample so as to be in the linear range Note The most probable list of causes is under each problem section Causes Solutions may overlap with other problems BioVision Incorporated Tel 408 493 1800 Fax 408 493 1801 155 S Milpitas Boulevard Milpitas CA 95035 USA www biovision com tech biovision com Page 2 of 2
7. y Sample Preparations Tissues 50 mg or cells 1 x 10 can be homogenized in 200 ul ice cold ALT Assay Buffer then centrifuged 13 000 x g 10 min to remove insoluble material BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA For research use only rev 1 15 Serum samples can be directly diluted in the Assay Buffer Prepare test samples of up to 20 ul 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 Reaction Mix Mix enough reagents for the number of assays to be performed For each well prepare a total 100 ul Reaction Mix ALT Assay Buffer 86 ul OxiRed Probe 2 ul ALT Enzyme Mix 2 ul ALT Substrate 10 ul Add 100 ul 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 ul of the probe per reaction to decrease the background reading amp increase detection sensitivity significantly Measurement Read OD 570 nm A at T T4 gt 10min then again Az at T 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 AA570 nm Ao Aj It is recommended that the user run the assay kinetically to choose A and A2 values which occur a
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