Glutathione Colorimetric Detection Kit - B
Contents
1. 70 C or analyzed immediately At this point the SSA concentration will be 2 5 The supernatant must be diluted 1 2 5 with Assay Buffer by mixing one part with 1 5 parts of Assay Buffer to bring the SSA concentration to 1 The sample will have been diluted 1 5 at this point All final dilutions are made in Sample Diluent Treated Whole Blood must be further diluted at least 1 20 for a recommended final dilution of 2 1 100 For Treated Plasma and Treated Urine a final dilution of gt 1 5 is recommended but further dilutions in Sample Diluent may be necessary Tissue Samples Fresh tissue is washed with ice cold PBS to remove blood then blotted on filter paper before recording wet weight NOTE Samples that have been frozen will contain lysed cells The PBS wash may contain substantial amounts of GSH and or GSSG e For Samples Where a Protein Determination is to be Obtained Homogenize at 10 mg 250 uL in ice cold 100mM phosphate buffer pH 7 Centrifuge at 14 000 rom for 10 minutes at 4 C and remove an aliquot of the supernatant for protein determination Thoroughly mix a second aliquot of the supernatant with an equal volume of cold 5 SSA Incubate for 10 minutes at 4 C Centrifuge at 14 000 rpm for 10 minutes at 4 C to remove precipitated protein Collect the supernatant The supernatant must be diluted 1 2 5 with Assay Buffer by mixing one part with 1 5 parts of Assay Buffer The SSA concentration will be 1 e For Samples Not Requir2. 0x106 cells mL in cold 5 SSA we used Jurkats at 5x106 cells mL and are lysed and deproteinized by vigorous vortexing freeze thaw cycling or other suitable disruption method Incubate cells at 4 C for 10 minutes followed by centrifugation for 10 minutes at 14 000 rpm and 4 C NOTE Samples that have been frozen will contain lysed cells The PBS wash may contain substantial amounts of GSH and or GSSG The deproteinized supernatants must be diluted 1 5 with Assay Buffer by mixing one part with 4 parts of Assay Buffer The SSA concentration will be 1 The sample will have been diluted 1 5 at this point Further sample dilutions must be done in Sample Diluent and need to be determined by the end user since it will be dependent upon the cell type and number of cells used The recommended final dilution is gt 1 20 Use all samples within 2 hours of dilution ASSAY PROTOCOL END POINT For Oxidized Glutathione GSSG use the 2VP treated standards 2VP treated Sample Diluent and 2VP treated samples diluted with Sample Diluent as described previously For Total Glutathione use the standards and samples diluted with Sample Diluent as described previously Pipet 50 uL of either 2VP treated or untreated samples or standards into duplicate wells in the plate Pipet 50 uL of either 2VP treated or untreated Sample Diluent into duplicate wells as the Zero Standard Add 25 uL of the Colorimetric Detection Reagent to each well using a repeater or multi
3. B Bridge International Inc kA Glutathione Colorimetric Detection Kit User Manual Catalog K3006 C B Bridge International Inc Glutathione 110628 TABLE OF CONTENTS Intended Use Background Assay Principle Kit Components Materials Required Precautions Reagent Preparation Sample Preparation Assay Protocol End Point Assay Protocol Kinetic Calculations CO GO NN OO F amp F Ff FW WB W Typical Standard Curve Example Notice to Purchaser This product is to be used for Research Purposes Only It is not to be used for Drug or Diagnostic Purposes nor is it intended for Human Use B Bridge products may not be resold modified for resale or used to manufacture commercial products without the express written consent of B Bridge International Inc EXCEPT AS OTHERWISE EXPRESSLY SET FORTH IN THIS USER MANUAL B BRIDGE DOES NOT MAKE ANY REPRESENTATION OR WARRANTIES OR CONDITIONS OF ANY KIND EITHER EXPRESSED OR IMPLIED WITH RESPECT TO THE PRODUCTS OR INFORMATION DISCLOSED HEREUNDER INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FIT FOR A PARTICULAR PURPOSE OR NONINFRINGEMENT OF THE INTELLECTUAL PROPERTY RIGHTS OF THIRD PARTIES B Bridge International Inc All Rights Reserved 2 B Bridge International Inc Glutathione 110628 INTENDED USE The B Bridge Glutathione Colorimetric Detection Kit cat K3006 C quantitatively measures Glutathione GSH and oxidized glutathione GSSG in Hum
4. an Whole Blood Serum Plasma Erythrocytes Urine Cell Lysates and Tissue Samples GSH is identical across species and we expect this kit may measure GSH from sources other than human BACKGROUND Glutathione L y glutamyl L cysteinylglycine GSH is the highest concentration non protein thiol in mammalian cells and is present in concentrations of 0 5 10 mM GSH plays a key role in many biological processes including the synthesis of proteins and DNA the transport of amino acids and the protection of cells against oxidation Harmful hydrogen peroxide cellular levels are minimized by the enzyme glutathione peroxidase GP using GSH as a reductant STAM The oxidized GSH dimer GSSG is formed from GSH and peroxide by the GP reaction see below An important role of GSSG in the NFkB activating signal cascade is suggested by the facts that the potent NFKB inducer tetradecanoyl phorbol acetate increases intracellular GSSG levels and GSSG GSH ratios H 0 H202 Glutathione Peroxidase GSSG GSH Glutathione eon S Transfemse NADPH NADP GSH Conjugate Glutathione S transferases GST are an important group of enzymes that catalyze the nucleophilic addition of GSH to electrophiles They are encoded by 5 gene families 4 encode cytosolic GST and one encodes the microsomal form of GST They have been implicated in a number of diseases In asthma arachidonic acid is converted to unstable leukotriene A LTA4 LTA is either hyd
5. card remaining unused solutions by mixing with copious amounts of water Dimethyl sulfoxide is a powerful aprotic organic solvent that has been shown to enhance the rate of skin absorption of skin permeable substances Wear protective gloves when using the solvent especially when it contains dissolved chemicals In all cases please consult your institution s safety procedures for working with hazardous chemicals B Bridge International Inc Glutathione 110628 REAGENT PREPARATION Allow the kit reagents to come to room temperature for 30 minutes We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine GSH concentrations Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit Sample Diluent Prepare the Sample Diluent by diluting one part 5 SSA 1 5 with four parts Assay Buffer and vortex thoroughly The pH of the Sample Diluent must be gt 6 Sample Diluent can be stored at 4 for one month 2 Vinylpyridine Treatment To measure Oxidized Glutathione free GSH must be blocked by alkylation To 250 uL of SSA treated samples standards or Sample Diluent add 5 uL of the ethanolic solution of 2VP see page 4 and allowed to incubate at room temperature for 1 hour The 2VP treated samples and standards should then be diluted in Assay Buffer and Sample Diluent according to the dilutions recommended for each sample type prio
6. channel pipet Add 25 uL of the Reaction Mixture to each of the wells using a repeater or multichannel pipet Gently tap the sides of the plate to ensure adequate mixing of the reagents Incubate at room temperature for 20 minutes N Oo fF GON Read the optical density at 405 nm These data will be used to determine either Oxidized Glutathione or Total Glutathione concentration ASSAY PROTOCOL KINETIC 1 Carry out steps 1 3 above 2 Gently tap the sides of the plate to ensure adequate mixing of the reagents 3 Add 25 uL of the Reaction Mixture to each of the wells using a repeater and immediately place plate in reader and read optical density at 405 nm every minute for at least 10 minutes These data will be used to determine Total or Oxidized Glutathione concentration kinetically B Bridge International Inc Glutathione 110628 CALCULATIONS Average the duplicate optical density readings for each standard and sample Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader after subtracting the mean ODs for the zero standard The concentrations obtained should be multiplied by the dilution factor to obtain sample values Glutathione concentrations see below are calculated from the data using the curve fitting routine supplied with the plate reader Oxidized Glutathione concentrations of the samples are determined from the data obtained from 2VP treated samples read off a 2VP
7. in the same well without using 2 Vinylpyridine B Bridge International Inc Glutathione 110628 KIT COMPONENTS Clear 96 well plate 4 plates Oxidized Glutathione Standard 250 uM 200 uL Detection Reagent Concentrate 1 mL Assay Buffer 225 mL NADPH Concentrate 1 mL Glutathione Reductase Concentrate 1mL Store above components at 4 C MATERIALS REQUIRED BUT NOT SUPPLIED Deionized or distilled water Aqueous 5 sulfo salicylic acid dihydrate SSA solution at 5 weight volume 1g of SSA per 20 mL of water for treating samples to remove protein 2 Vinylpyridine 2VP is used to block any free GSH or other thiols present in the treated samples 2VP is prepared by adding 27 uL of 2 vinylpyridine to 98 uL of ethanol Use immediately and discard remaining unused solutions A 96 well plate reader capable of reading optical absorption at 405 412 nm Software for converting raw optical density readings from the plate reader and carrying out four parameter logistic curve 4PLC fitting Contact your plate reader manufacturer for details PRECAUTIONS This kit should only be used by qualified personnel who have had laboratory safety instruction The complete User Manual should be read and understood before using this product Sulfosalicylic acid is a strong acid solution and should be treated like any other laboratory acid 2VP is TOXIC and may cause burns 2VP solutions should be prepared in a fume hood Use im mediately and dis
8. ing a Protein Determination Homogenize at 10 mg 250 uL in ice cold 5 SSA incubate at 10 minutes at 4 C then centrifuge at 14 000 rpm for 10 minutes at 4 C to remove precipitated B Bridge International Inc Glutathione 110628 protein Collect the supernatant The supernatant must be diluted 1 5 with Assay Buffer by mixing one part with 4 parts of Assay Buffer The SSA concentration will be 1 Further sample dilutions must be determined by the end user since it will be dependent upon the tissue type and the amount of tissue used These dilutions must be made in the prepared Sample Diluent Erythrocytes Red Blood Cells RBC s Collect blood with heparin or EDTA Centrifuge the sample remove and discard the plasma and white cell layer Wash the RBC s 2 times by suspending in 3 volumes of isotonic saline 0 9 centrifuging at 600 x g for 10 minutes and discarding the saline wash After the 2 washes mix 250uL RBC s with 1mL of cold 5 SSA Incubate for 10 minutes at 4 C and centrifuge at 14 000 rpm for 10 minutes at 4 C Collect the supernatant At this point the SSA concentration will be 4 The supernatant must be diluted 1 4 with Assay Buffer by mixing one part with 3 parts of Assay Buffer The SSA concentration will now be 1 and the sample will have been diluted 1 20 at this point Further dilutions are made in Sample Diluent for a recommended final dilution of 1 40 Cell Lysates Washed cell pellets are resuspended at 1 1
9. lutathione Standard should be treated with 1 uL of2VP as outlined on page 9 2VP treated Standards are prepared by labeling six test tubes as 1 through 6 Pipet 475 uL of Sample Diluent into tube 1 and 250 uL into tubes 2 to 6 Carefully add 25 uL of the 2VP treated Standard to tube 1 and vortex completely Take 250 uL of the solution in tube 1 and add it to tube 2 and vortex completely Repeat for tubes 3 through 6 as indicated in the table below The concentration of Oxidized Glutathione in tubes 1 through 6 will be 12 5 6 25 3 125 1 56 0 781 and 0 391 uM 2VP treated Sample Diluent must be used as a 0 uM standard To Determine Total GSH Standards are prepared by labeling six test tubes as 1 through 6 Pipet 475 uL of Sample Diluent into tube 1 and 250 uL into tubes 2 to 6 Carefully add 25 uL of the supplied Standard to tube 1 and vortex completely Take 250 uL of the solution in tube 1 and add it to tube 2 and vortex completely Repeat for 5 B Bridge International Inc Glutathione 110628 tubes 3 through 6 according to the table below The concentration of Total GSH in tubes 1 through 6 will be 25 12 5 6 25 3 125 1 56 and 0 781 uM after addition of the Reaction Mixture Sample Diluent must be used as a 0 uM standard Standard Standard Standard Standard Standard Standard Reagent a DR 3 4 ANSE ns eee Sample Diluent 475 ul 250 ul 250 ul 250 ul 250 ul 250 ul Gl
10. r to using in the assay The 2VP treated Sample Diluent is used for the zero standard Samples treated with 2VP should be read off a standard curve generated with 2VP treated standards Colorimetric Detection Reagent Prepare the Colorimetric Detection Reagent by diluting one part Colorimetric Detection Reagent Concentrate 1 10 with nine parts Assay Buffer See Colorimetric Detection Reagent Dilution Table for suitable volumes Colorimetric Detection Reagent Dilution Table Whole Two Four Reagent Half plate plate Plates Plates Colorimetric Detection Concentrate 140 ul 260 ul 500 ul 1 mL Assay Buffer 1 26 mL 2 34 mL 4 5 mL 9 mL Total Colorimetric Reagent Volume 1 4 mL 1 6 mL 5 mL 10 mL Reaction Mixture Prepare the Reaction Mixture by diluting one part each NADPH and Glutathione Reductase Concentrates 1 10 into eight parts Assay Buffer See Reaction Mix Dilution Table for suitable volumes Store any unused Reaction Mixture at 4 C for no more than 2 days Reaction Mix Dilution Table Whole Two Four Reagent Half plate plate Plates Plates NADPH Concentrate 140 ul 260 ul 500 ul 1 mL Glutathione Reductase Concentrate 140 ul 260 ul 500 ul 1 mL Assay Buffer 1 12 mL 2 08 mL 4mL 8 mL Total Reaction Mix Volume 1 4 mL 1 6 mL 5 mL 10 mL Standard Preparation To Determine GSSG For the measurement of Oxidized Glutathione GSSG a 50 uL aliquot of the 250 uM Oxidized G
11. rated to form LTB or it is conjugated to GSH by a GST leukotriene C synthase to form leukotriene Cy LTC and its derivative LTD are important molecules in bronchial asthma Leukotriene C synthase is therefore an important therapeutic target It has also been shown that increased expression of GSTs can lead to drug resistance Three glutathione adducts of the drug melphalan used to treat ovarian cancer and multiple myeloma have been isolated from reactions involving human microsomal GSTs ASSAY PRINCIPLE The Glutathione Colorimetric Detection Kit is designed to quantitatively measure glutathione GSH and oxidized glutathione GSSG present in a variety of samples The kit utilizes a colorimetric substrate that reacts with the free thiol group on GSH to yield a highly colored product Supplied reagents are in solution and require simple dilution for use in the assay By using 2 Vinylpyridine not supplied to block any free GSH in the sample Oxidized Glutathione GSSG can be determined Any samples that have not been treated with 2 Vinylpyridine will yield Total GSH levels The Free GSH concentration in the sample is calculated from the difference between the Total GSH determined and the GSH generated from Oxidized Glutathione for the 2 Vinylpyridine treated samples Our Fluorescent Glutathione Detection kit Catalog Number K3006 1 allows for the measurement of both Free and Oxidized Glutathione with higher sensitivity in the same sample
12. treated standard curve The concentration of Oxidized Glutathione GSSG in the samples would be half of the GSH concentration read off the curve Note 1 GSSG 2 GSH Free glutathione GSH concentrations are obtained by subtracting the Oxidized Glutathione GSSG levels obtained from the 2VP treated standard and samples from non treated standards and samples Total GSH Concentrations obtained will be in uM of Glutathione Total GSH Free GSH Oxidized GSH GSSG Oxidized GSH measured 2VP Treated GSH concentration 2 Free GSH Total GSH Conc Oxidized GSH Conc TYPICAL STANDARD CURVE EXAMPLE ONLY Net OD Total Glutathione Conc uM Always run your own standard curves for calculation of results Do not use this data B Bridge International Inc Glutathione 110628
13. utathione Standard 25 ul Standard 1 250 ul Standard 2 250 ul Standard 3 250 ul Standard 4 250 ul Standard 5 250 ul GSSG Concentration 12 5 uM 6 25 uM 3 125 uM 1 56 uM 0 781 uM 0 391 uM Total GSH Concentration 25 uM 12 5 uM 6 25 uM 3 125 uM 1 56 uM 0 781 uM Use all Standards within 2 hours of preparation SAMPLE PREPARATION Sample Diluent Prepare the Sample Diluent by diluting one part 5 SSA 1 5 with four parts Assay Buffer and vortex thoroughly The pH of the Sample Diluent must be gt 6 Sample Diluent can be stored at 4 C for one month All samples and standards must be in Sample Diluent before starting the assay To measure Oxidized Glutathione in samples reduced Glutathione GSH in the sample must be blocked by treatment with 2 vinylpyridine 2VP see page 6 for preparation SSA treated samples should be treated with 2VP by addition of 5 uL of 2VP solution for every 250 uL of sample see page 9 2VP treated samples must be read off a standard curve made with 2VP treated standards Use all samples within 2 hours of dilution Whole Blood EDTA or Heparin Plasma or Urine Thoroughly mix sample with an equal volume of cold 5 SSA Incubate for 10 minutes at 4 C Centrifuge at 14 000 rpm for 10 minutes at 4 C Collect the supernatant If the supernatent contains particulates re centrifuge the supernatant for 15 minutes and collect the clarified second supernatant Samples can be stored in aliquots at 2
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