Data Sheet - BioVision
Contents
1. BioVision ApoGSH Glutathione Colorimetric Detection Kit Catalog K261 100 100 assays Store kit at 20 C Introduction Glutathione GSH is the major intracellular low molecular weight thiol that plays a critical role in the cellular defense against oxidative stress in mammalian cells BioVision s ApoGSH Glutathione Colorimetric Assay Kit provides a convenient colorimetric method for analyzing either total glutathione or the reduced form glutathione alone using a microtiter plate reader The assay is based on the glutathione recycling system by DTNB and glutathione reductase Fig 1 DTNB and glutathione GSH react to generate 2 nitro 5 thiobenzoic acid which has yellow color Therefore GSH concentration can be determined by measuring absorbance at 412 nm The generated GSSG can be reduced back to GSH by glutathione reductase and GSH reacts with DTNB again to produce more 2 nitro 5 thiobenzoic acid Therefore the recycling system dramatically improves the sensitivity of total glutathione detection The kit includes the 5 Sulfosalicylic acid SSA for the removal of proteins from samples and for the protection of GSH oxidation and y glutamyl transpeptidase reaction The kit can quantify glutathione from 1 100 ng well in a 200 Ol reaction For detecting lower glutathione concentrations such as in blood samples increasing reaction time will generate stronger signal The kit can also specifically detect the reduced form of gluta2. Use fresh components from the same kit Unanticipated results 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 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 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 For research use only Page 3 of 3
3. from the remaining sample solution after the plasma sample isolation Preparation of Solutions amp Storage Conditions Substrate Add 1 ml of Glutathione Buffer to 1 vial of substrate and dissolve it completely Store the remaining solution at 20 C stable for 2 months NADPH Generating Mix Add 1 ml of Glutathione Buffer to 1 vial of the NADPH mix Store the solution at 20 C stable for 2 months Glutathione Reductase Add 1 ml of Glutathione Buffer to 1 vial of the enzyme and dissolve Use up the solution within 1 day SSA Add 19 ml of dH20 to make 5 solution and then dilute 5 ml of the solution with Glutathione Buffer to make 1 SSA solution Store at 4 C stable for 6 months GSH Standard Add 1 ml of 1 SSA to the GSH standard vial to generate 1ug ul GSH standard solution Store at 20 C stable for 2 months Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 3 BioVision V Vi 1 Vil Vill Preparation of Solutions for Standard Curve To generate standard curve for detecting the reduced form of glutathione only add 50 40 30 20 10 and O ul of the 1 g l GSH standard into each labeled microcentrifuge tubes add 1 SSA to make up for a total volume of 100 ul tube To generate standard curve for detecting the total glutathione dilute the 1 ug l glutathione solution into 10 ng pl with 1 SSA Add 50 40 30 20 10 and 0 ul of the 10 ng ul GSH standard int
4. l Cholesterol Quantification Kit Molecular Biology amp Reporter Assays e siRNA Vectors e Cloning Insert Quick Screening Kit e Mitochondrial amp Genomic DNA Isolation Kits 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 2 of 3 BioVision GENERAL TROUBLESHOOTING GUIDE rev 08 12 Problems Cause Solution Assay not working e Use of ice cold reaction buffer e Omission of a step in the protocol e Plate read at incorrect wavelength e Use of a different 96 well plate e Reaction buffer must be at room temperature 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 Luminescence White plates Colorimeters Clear plates Samples with erratic readings e Use of an incompatible sample type e Samples prepared in a different buffer e Samples were not deproteinized if indicated in datasheet 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 Refer data sheet for details about incompatible samples e Use the reaction buffer provided in the kit or refer data sheet for instructions e Use the 10 kDa spin cut off filter or PCA precipitation as indicated e Use Dounce homogeni
5. o each labeled microcentrifuge tubes add 1 SSA to make up for a total volume of 100 il tube Glutathione Assay Protocol Prepare enough Reaction Mix for the standard and samples to be assayed in 96 well plate not provided Each well should contain 20 ul NADPH Generating Mix 20 ul Glutathione Reductase 120 ul Glutathione Reaction Buffer For detecting the reduced form of glutathione only omit Glutathione Reductase Use 20 ul of the Glutathione Reaction Buffer replace the 20 ul of glutahione Reductase Mix well Add 160 ul of the Reaction Mix to each well and incubate at room temperature for 10 minutes to generate NADPH Add 20 ul of either the GSH standard solutions or the sample solution Incubate the plate at room temperature for 5 10 min Note We recommend to make several dilutions of your sample using the 1 SSA to make sure the readings are within the range of the standard calibration curve Add 20 ul of Substrate solution and incubate at room temperature for 5 10 min or longer if the samples contain low levels of glutathione Notes a Since the reaction starts immediately after the addition of substrate use a multichannel pipette or repeating pipette is recommended to avoid the reaction time lag among wells b You can read samples immediately and at various times following addition of the substrate solution for kinetic studies Read the absorbance at 405 nm or 415 nm using a microplate reader Determine concentra
6. rnatant 3 Resuspend cell pellet in 0 5 ml ice cold PBS Transfer into a 1 5 ml microcentrifuge tube and centrifuge at 700 x g for 5 minutes at 4 C Remove supernatant 4 Lyse cells in 80 ul ice cold Glutathione Buffer Incubate on ice for 10 minutes 5 Add 20 ul of 5 SSA see below for SSA preparation mix well and centrifuge at 8000 x g for 10 min Transfer supernatant to a fresh tube and use it for glutathione assay B Tissue Sample Preparation 100 mg 1 Homogenize the tissue in 0 4 ml of Glutathione Buffer 2 Add 100 ul of 5 SSA see below for SSA preparation mix well and centrifuge at 8000 x g for 10 minutes 3 Transfer supernatant to a fresh tube and use it for glutathione assay C Plasma Sample Preparation 1 Centrifuge an anticoagulant treated blood at 1000 x g for 10 min at 4 C 2 Transfer the top plasma layer to a new tube and add 1 4 vol of 5 SSA Mix well 3 Centrifuge at 8000 x g for 10 min at 4 C 3 Transfer supernatant to a new tube and use it for the glutathione assay D Erythrocyte Sample Preparation 1 Centrifuge an anticoagulant treated blood at 1000 x g for 10 min at 4 C 2 Discard the supernatant and the white buffy layer 3 Lyse the erythrocytes with 4 vol of Glutathione Buffer Keep on ice for 10 min 4 Add 1 vol 5 SSA mix well and centrifuge at 8000 x g for 10 minutes Transfer supernatant to a fresh tube and use it for glutathione assay Note Erythrocytes can be isolated
7. thione GSH by omitting the glutathione reductase from the reaction mixture The sensitivity for detecting the reduced form of glutathione without recycling system is 100 times lower than detecting the total glutathione NO 2GSH x HOOC Ss boai S ONI DTNB Glutathione A reductase HOOG _S 2 i GSSG ON 2 Nitro 5 thiobenzoic acid Fig 1 Principle of Total Glutathione Assay Kit Contents 100 ml NM K261 100 1 2 vials Red K261 100 2 2 vials Blue K261 100 3 2 vials Green K261 100 4 1 bottle WM K261 100 5 2x1mg Yellow K261 100 6 Component Glutathione Reaction Buffer Glutathione Substrate DTNB NADPH Generating Mix lyophilized Glutathione Reductase lyophilized Sulfosalicylic Acid SSA 1 gram GSH Standard lyophilized MW 307 BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA For research use only rev 08 12 Sample Preparation Note Peptide thiol may interfere with the assay of reduced form glutathione SSA treatment may not able to complete remove all small peptides from samples Further purification may be required to accurately measure reduced form glutathione Peptide thiols don t significantly interfere with total glutathione assay A Cell Sample Preparation 0 5 1 x 10 cells assay 1 Treat cells by desired method Concurrently incubate a control culture without treatment 2 Collect cells by centrifugation at 700 x g for 5 minutes at 4 C Remove supe
8. tions of GSH in the sample solutions using the standard glutathione calibration curve Note A Using reduced form glutathione Standard Curve for detecting reduced form of glutathione Using total Glutathione Standard Curve for detecting total glutathione There are about 10 to 100 fold difference in detection sensitivity between detecting reduced form glutathione and total glutathione see procedure step IV for preparation of standard curve B The colorimetric reaction is stable and the O D increases linearly over 30 min for total glutathione detection Calculation of Total Glutathione Pseudo end point method Kinetic method Total Glutathione O D sampie O D biank Total Glutathione Slopesampie SlOpepiank Slope Reagent Interference Reducing agents such as ascorbic acid B mercaptoethanol dithiothreitol DTT and cysteine or thiol reactive compounds such as maleimide compounds interfere with the glutathione assay and therefore should be avoided during the sample preparation BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA For research use only When detecting the reduced form of glutathione protein thiols can generate significant background signal In such cases it is necessary to completely remove proteins from samples We suggest using Centrifugal Spin column with 10 kDa molecular weight cut off filter BioVision Cat No 1997 25 to remove proteins Then the reduced glutathione can be easil
9. y detected from spin through samples rev 08 12 2 2 5 1 6 2 E E 12 g 15 8 08 al d fo Q 0 4 0 5 0 0 0 2 4 6 8 10 0 20 40 60 80 100 Reduced Glutathione ug Total Glutathione ng Fig 2 Glutathione Standard Curve Various amounts of standard glutathione was added to the glutathione reaction and incubated for 10 min according to the kit instructions Absorbance was measured at O D 405 nm 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 e Nuclear Apoptosis Kits amp Reagents e Apoptosis siRNA Vectors 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 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 Fluorometric amp Colorimetric Assay Kits e 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 Tota
10. zer 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 deproteinize samples e Use fresh samples or store at correct temperatures till use Lower Higher readings in Samples and Standards 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 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 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 Readings do not follow a linear pattern for Standard curve 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 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
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