data Sheet - BioVision
Contents
1. 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 155 S Milpitas Boulevard Milpitas CA 95035 USA Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 3 of 32. 1801 www biovision com tech biovision com Page 2 of 3 BioVision GENERAL TROUBLESHOOTING GUIDE rev 3 15 For research use only Problems Cause Solution Assay not working Samples with erratic readings Lower Higher readings in Samples and Standards Readings do not follow a linear pattern for Standard curve Unanticipated results e Use of ice cold assay buffer e Omission of a step in the protocol e Plate read at incorrect wavelength e Use of a different 96 well plate e Use of an incompatible sample type e Samples prepared in a different buffer e Cell tissue samples were not completely homogenized e Samples used after multiple free thaw cycles e Presence of interfering substance in the sample e Use of old or inappropriately stored samples e Improperly thawed components e Use of expired kit or improperly stored reagents e Allowing the reagents to sit for extended times on ice e Incorrect incubation times or temperatures Incorrect volumes used e Use of partially thawed components e Pipetting errors in the standard e Pipetting errors in the reaction mix e Air bubbles formed in well e Standard stock is at an incorrect concentration e Calculation errors e Substituting reagents from older kits lots e Measured at incorrect wavelength e Samples contain interfering substances e Use of incompatible sample type e Sample readings above below the linear range e Assay buffer must be at room tempera
3. BioVision GST Colorimetric Activity Assay Kit Catalog K263 100 100 assays Store kit at 20 C Introduction Glutathione S transferase GST is a family of enzymes that plays an important role in detoxification of xenobiotics GST catalyzes attachment of the thiol of glutathione to electrophiles Glutathione is used to scavenge potentially toxic compounds including those produced as a result of oxidative stress and is part of the defense mechanism neutralizing the mutagenic carcinogenic and toxic effects of such compounds The GST Colorimetric Activity Assay Kit is based upon the GST catalyzed reaction between GSH and the GST substrate CDNB 1 chloro 2 4 dinitrobenzene which has the broadest range of isozyme detectability e g alpha mu pi and other GST isoforms except theta Under certain conditions the interaction between glutathione and CDNB is totally dependent on the presence of active GST GST Oz Os The GST catalyzed formation of GS DNB produces a dinitropheny thioether which can be detected by spectrophotometry at 340 nm One unit of GST activity is defined as the amount of enzyme producing 1 umol of GS DNB conjugate min under the conditions of the assay The kit can detect GST activity in crude cell lysate or purified protein fractions and can quantitate GST tagged fusion proteins Detect limit Active GST lt 1mU Kit Contents K263 100 Cap Code Part Number GST Assay Buffer K263 100 1 GST Substrate CD
4. NB K263 100 2 Glutathione GSH lyophilized K263 100 3 GST Positive Control K263 100 4 Reagent Preparation and Storage Conditions GST Assay Buffer store at 4 C GSH Add 275 ul of GST Assay Buffer to each vial just before use One vial is sufficient for 50 assays The Remaining solution can be kept at 20 C for 1 week CDNB This vial contains a DMSO solution of 1 chloro 2 4 dinitrobenzene CDNB and should be stored at 20 C GST Positive Control Store at 20 C Sample Preparation Guideline Cell Sample Preparation 1 Collect cells by centrifugation For adherent cells use a rubber policeman to scrape and collect the cells 2 Homogenize or sonicate the cells in GST Assay Buffer typically 3 4 volumes 3 Centrifuge at 10 000 x g for 15 min at 4 C 4 Collect supernatant and use for the assay The remaining sample should be stored at 80 C and is stable for at least 1 month Tissue Sample Preparation 1 Prior to dissection perfuse tissue with PBS containing heparin 0 15 mg ml to remove red blood cells and clots 2 Homogenize tissue in GST Assay Buffer 100 mg 0 5 ml 3 Centrifuge at 10 000 x g for 15 minutes at 4 C 4 Collect supernatant and use for the assay The remaining sample should be stored at 80 C and is stable for at least 1 month Plasma and Erythrocyte Sample Preparation 1 Centrifuge anticoagulant treated blood at 1000 x g for 10 min at 4 C BioVision Incorpora
5. just the final volume to 50 ul with GST Assay Buffer Note We recommend preparing several dilutions of your sample and running duplicate wells for each measurement 2 Glutathione Addition Add 5 ul of Glutathione to each well containing the sample or control above 3 Substrate Mix Mix enough reagents for the number of assays to be performed For each well prepare a total 50 ul Substrate Mix containing GST Assay Buffer 49 ul GST Substrate CDNB Solution 1 ul Mix well and transfer 50 ul of the Mix into each sample including the standard well 4 Measurement Carefully shake the plate to start the reaction Read the absorbance once every minute at 340 nm using a plate reader to obtain at least 5 time points For low GST activity samples the reaction can be continued for longer time periods 0 6 0 5 y 0 1101x 0 0155 R2 0 9964 E 0 4 S 0 3 Activity 0 036 x O 1016 x 100 0 002 2 198 U ml 0 2 See calculation method below 0 1 0 0 1 2 3 4 5 Time min Figure GST Kinetic Assay Performed According to This Protocol Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 3 Bio Vision 3 Calculation of GST Assay Results Determine the change in absorbance AA340 per minute by i Plotting the absorbance values as a function of time to obtain the slope rate of the linear portion of the curve li Select two points on the linear portion of the curve and determine the cha
6. nge in absorbance during that time using the following equation A340 Time 2 A340 Time 1 AA340 min Time 2 min Time 1 min Determine the rate of AA340 min for the background wells and subtract the rate from that of the sample wells Use the following formula to calculate the GST activity U ml of sample The reaction rate at 340 nm can be determined using the GS DNB extinction coefficient at 340 nm 0 0096 Mcm The value has been adjusted for the path length of the solution in the well 0 2893 cm AAzaomin x Reaction Vol GST Activity ssomin x eaction Volume ml XD 0 0096 umol cm x 1000 ml x 0 2893 cm x V AAzmin x 0 036x D V umol min ml Where 0 0096 umol cm is the extinction coefficient of the glutathione DNB adduct V Sample Volume added to well ml D Sample Dilution Factor 0 2893 cm is light path of the 0 1 ml Reaction Volume in a Greiner Bio One 655101 96 well plate cm Other plates must be calibrated for accurate results Unit Definition One unit is the amount of enzyme that conjugates 1 0 umol of 1 Chloro 2 4 Dinitrobenzene with reduced glutathione per min at pH 6 4 at 25 C BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 3 15 For research use only RELATED PRODUCTS Apoptosis Detection Kits amp Reagents e Annexin V Kits amp Bulk Reagents e Caspase Assay Kits amp Reagents e Mitochondrial Apoptosis Kits amp Reagents Nuclear Ap
7. optosis Kits amp Reagents Cell Fractionation System e _Mitochondria Cytosol Fractionation Kit Nuclear Cytosol Fractionation Kit Membrane Protein Extraction Kit Cytosol Particulate Rapid Separation Kit Mammalian Cell Extraction Kit e FractionPREP Fractionation System Cell Proliferation amp Senescence e Quick Cell Proliferation Assay Kit Senescence Detection Kit High Throughput Apoptosis Cell Viability Assay Kits LDH Cytotoxicity Assay Kit Bioluminescence Cytotoxicity Assay Kit e Live Dead Cell Staining Kit Cell Damage amp Repair e HDAC Fluorometric amp Colorimetric Assays amp Drug Discovery Kits e HAT Colorimetric Assay Kit amp Reagents e DNA Damage Quantification Kit e Glutathione amp Nitric Oxide Fluorometric amp Colorimetric Assay Kits Signal Transduction e cAMP amp cGMP Assay Kits e Akt amp JNK Activity Assay Kits e Beta Secretase Activity Assay Kit Adipocyte amp Lipid Transfer e Recombinant Adiponectin Survivin amp Leptin e CETP Activity Assay amp Drug Discovery Kits e Total Cholesterol Quantification Kit Molecular Biology amp Reporter Assays e siRNA Vectors e Cloning Insert Quick Screening Kit e Mitochondrial amp Genomic DNA Isolation Kits e 5 Minutes DNA Ligation Kit e 20 Minutes Gel Staining Destaining Kit Growth Factors and Cytokines Monoclonal and Polyclonal Antibodies and Cytokines FOR RESEARCH USE ONLY Not to be used in humans Tel 408 493 1800 Fax 408 493
8. ted 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 3 15 For research use only 2 Transfer the top plasma layer without disturbing the white buffy layer to a new tube and store on ice for assay or store at 80 C for future use The plasma should be stable for 1 month 3 Remove the white buffy layer and discard leukocytes 4 Lyse the erythrocytes red blood cells in 4 times its volume of ice cold GST Assay Buffer 5 Centrifuge at 10 000 x g for 15 min at 4 C 6 Transfer supernatant erythrocyte lysate to a new tube and use it for the GST assay The remaining samples should be stored at 80 C for future use and is stable for at least one month Preparation of Bacterially Expressed GST Fusion Protein Sample 1 Collect bacteria by centrifugation Freeze thaw the pellet two times then sonicate in GST Assay Buffer 2 Centrifuge at 10 000 x g for 15 min at 4 C 3 Transfer supernatant to a new tube and use it for the GST assay The remaining samples should be stored at 80 C for future use and is stable for at least one month GST Assay Protocol 1 Sample Negative Control and Positive Control Preparation Prepare samples in a total 50 ul volume with GST Assay Buffer including a negative control with 50 ul of GST Assay buffer only For GST Positive Control dilute 100 time by adding 2 ul of Positive Control into 198 ul GST Assay Buffer add 2 10 ul of diluted GST Positive Control into desired well s and ad
9. ture e Refer and follow the data sheet precisely 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 Clear plates 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 e 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 e Thaw all components completely and mix gently before use e Always check the expiry date and store the components appropriately e 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 e Thaw and resuspend all components before preparing the reaction mix e Avoid pipetting small volumes e Prepare a master reaction mix whenever possible e 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 e Use fresh components from the same kit e 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
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