Data Sheet - BioVision
Contents
1. AST Activity nmol min ml mU ml B T2 T1 x V Where B is the glutamate amount calculated from the Standard Curve in nmol T is the time of the first reading A1 in min Tz is the time of the second reading Ag in min V is the original sample volume added into the reaction well in ml One unit of AST is defined as the amount of AST which generates 1 0 umol of glutamate per minute at 37 C Positive Control Glutamate Standard Curve Positive Control initial lag phase So 3 3 6 0 5 y 0 0781x 0 2062 R 0 9917 03 Background 0 a a a a a 0 5 10 0 10 20 30 40 50 60 Glutamate nmol Time min RELATED PRODUCTS Alanine Transaminase Assay Kit NADP NADPH Quantitation Kit ADP ATP Ratio Assay Kit Glutamate Dehydrogenase Kit Glucose Assay Kit Fatty Acid Assay Kit Pyruvate Kinase Assay Kit LDH Quantification Kit Pyruvate Assay Kit para Aminohippuric Acid Kit Triglyceride Assay Kit Lipase Assay Kit Glycogen Assay Kit Lactate assay Kit Glucose Assay Kit Creatinine Assay Kit Fatty Acid Assay Kit Cholesterol Assay Kit Sarcosine assay Kit Uric Acid assay Kit 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 BioVision GENERAL TROUBLESHOOTING GUIDE For research use only rev 02 13 Problems Cause Solution Assay not working e Use of ice cold assay buffer e Omission of a step in the p2. Always thaw and prepare fresh reaction mix before use e Refer datasheet amp verify correct incubation times and temperatures e Use calibrated pipettes and aliquot correctly Readings do not follow a linear pattern for Standard curve e Use of partially thawed components Pipetting errors in the standard Pipetting errors in the reaction mix Air bubbles formed in well Standard stock is at an incorrect concentration e Calculation errors e Substituting reagents from older kits lots Thaw and resuspend all components before preparing the reaction mix e Avoid pipetting small volumes Prepare a master reaction mix whenever possible Pipette gently against the wall of the tubes e Always refer the dilutions in the data sheet e Recheck calculations after referring the data sheet Use fresh components from the same kit Unanticipated results e Measured at incorrect wavelength Samples contain interfering substances e Use of incompatible sample type Sample readings above below the linear range Check the equipment and the filter setting e Troubleshoot if it interferes with the kit e Refer data sheet to check if sample is compatible with the kit or optimization is needed 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 155 S Mil
3. 100 2 AST Developer lyophilized 1 vial Red K753 100 3 AST Substrate lyophilized 1 vial Orange K753 100 4 Glutamate Standard 0 1M 0 1 ml Yellow K753 100 5 AST Positive Control lyophilized 1 vial Blue K753 100 6 lll Storage and Handling Store the kit at 20 C protected from light Allow the Assay Buffer to warm to room temperature before use Briefly centrifuge vials before opening Read the entire protocol before performing the assay IV Reagent Preparation AST Enzyme Mix Reconstitute with 220 ul dH20 Aliquot and store at 20 C Use within two months Developer Reconstitute with 820 ul dH20 Aliquot and store at 20 C Use within two months AST Substrate Reconstitute with 1 1 ml assay buffer Store at 20 C Use within two months AST Positive Control Reconstitute with 100 ul dH20 Aliquot and store at 20 C Use within two months In the assay optional add 5 ul positive control and adjust the volume to 50ul well with Assay Buffer V AST Assay Protocol 1 Standard Curve Preparation Dilute 10 ul of the 0 1M Glutamate Standard with 990 ul Assay Buffer to generate 1 mM glutamate Add 0 2 4 6 8 10 ul into each well individually Adjust the final volume to 50 ul well with Assay Buffer to generate 0 2 4 6 8 10 nmol well of Glutamate Standard 2 Sample Preparations Tissues 50 mg or cells 1 x 10 can be homogenized 200 ul of ice cold Assay Buffer then centrifuge 13 000 x g 10 min to remove
4. BioVision Aspartate Aminotransferase AST or SGOT Activity Colorimetric Assay Kit Catalog K753 100 100 assays Store kit at 20 C l Introduction Aspartate aminotransferase AST also known as Glutamate oxaloacetate transaminase GOT is a transaminase EC 2 6 1 1 similar to the more liver specific alanine transaminase ALT Although commonly included clinically as part of a diagnostic liver function test AST has a broader clinical utility since it may also be elevated in diseases affecting other organs such as the heart or muscles in myocardial infarction also in acute pancreatitis acute hemolytic anemia severe burns acute renal disease musculoskeletal diseases and trauma It catalyzes the reaction Aspartate 2l Ketoglutarate Oxaloacetate Glutamate Diagnostically it is almost always measured in units liter U I In BioVision s AST Assay Kit an amino group is transferred of from aspartate to f ketoglutarate The products of this reversible transamination reaction are oxaloacetate and glutamate The glutamate is detected in a reaction that concomitantly converts a nearly colorless probe to color Amax 450 nm The kit provides a rapid simple sensitive and reliable test suitable as a high throughput activity assay of AST with a detection limit of 10 mU per well Il Kit Contents Components 100 assays Cap Code Part Number AST Assay Buffer 25 ml WM K753 100 1 AST Enzyme Mix lyophilized 1 vial Green K753
5. insoluble material Serum samples can be directly diluted in the Assay Buffer Prepare test samples of up to 50 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 BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA For research use only rev 02 13 3 Reaction Mix Mix enough reagent for the number of assays to be performed For each well prepare a total 100 ul Reaction Mix AST Assay Buffer 80 ul AST Enzyme Mix 2 ul Developer 8 ul AST Substrate 10 ul Add 100 ul of the Reaction Mix to each well containing the Samples Standards and Positive Controls optional Mix well 1 Measurement Read OD 450 nm A at T T4 gt 10 min then again A2 at T after incubating the reaction at 37 C for 60 min or longer if the AST activity is low protect from light The OD of the color generated by deamination of glutamate is AA450 nm A2 Aj It is recommended that the user run the assay kinetically to choose A 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 generated in the standard curve 4 Calculation Plot the glutamate standard curve and use the AA450 nm to obtain B nmol of glutamate amount of glutamate generated between T and Tz in the reaction wells AST activity in the test samples can then be calculated
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7. rotocol e Plate read at incorrect wavelength e Use of a different 96 well plate e Assay buffer must be at room temperature e Refer and follow the data sheet precisely Check the wavelength in the data sheet and the filter settings of the instrument e Fluorescence Black plates clear bottoms Luminescence White plates Colorimeters Clear plates Samples with erratic readings e Use of an incompatible sample type Samples prepared in a different buffer Cell tissue samples were not completely homogenized Samples used after multiple free thaw cycles e Presence of interfering substance in the sample e Use of old or inappropriately stored samples e Refer data sheet for details about incompatible samples 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 Aliquot and freeze samples if needed to use multiple times e Troubleshoot if needed e Use fresh samples or store at correct temperatures until use Lower Higher readings in Samples and Standards Improperly thawed components Use of expired kit or improperly stored reagents Allowing the reagents to sit for extended times on ice e Incorrect incubation times or temperatures e Incorrect volumes used e Thaw all components completely and mix gently before use e Always check the expiry date and store the components appropriately
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