Mouse VEGF ELISA Kit User Manual Catalog
Contents
1. Working Solution Preparation Pour entire contents 30 ml of the Wash Buffer Concentrate into a clean 1 000 ml graduated cylinder Bring final volume to 600 ml with glass distilled or deionized water 1 20 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Vill ASSAY PROCEDURE The Streptavidin HRP Working Solution and TMB Substrate Solution must be kept warm at 37 C for 30 minutes before use When diluting samples and reagents they must be mixed completely and evenly Standard detection curve should be prepared for each experiment The user will decide sample dilution fold by crude estimation of protein amount in samples 1 Add 100 ul of each standard and sample into appropriate wells 2 Cover well and incubate for 90 minutes at room temperature or over night at 4 C with gentle shaking 3 Remove the cover discard the solution and wash plate 3 times with Wash Buffer Working Solution and each time let Wash Buffer Working Solution stay in the wells for 1 2 minutes Blot the plate onto paper towels or other absorbent material Do NOT let the wells completely dry at any time 4 Add 100 ul of Biotin Labeled Detection Antibody Working Solution into each well and incubate the plate at 37 C for 60 minutes 5 Wash plate 3 times with Wash Buffer Working Solution and each time let Wash Buffer Working Solution stay in the wells for 1 2 minutes Discard the Wash Buffer Working Solution and blot the plate o2. with Wash Buffer Working Solution Add 100 ul Biotin Labeled Detection Antibody Working Solution Wash plate 3 times with Wash Buffer Working Solution Add 100 ul Streptavidin HRP Working Solution Wash plate 5 times with Wash Buffer Working Solution Add 100 ul TMB Substrate Solution Add 100 ul Stop Solution f Read the plate at 450nm FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES X TYPICAL DATA The standard curve is for demonstration only A standard curve must be run with each assay 10 g 1 5 3 0 01 1 L 1 4 1 L 1 1 1 L 1 1 1 10 100 1 000 Mouse VEGF Concentration pg ml XI SENSITIVITY The minimum detectable dose of Mouse VEGF is typically less than 2 pg ml XII SPECIFICITY The Mouse VEGF ELISA Kit allows for the detection and quantification of endogenous levels of natural and or recombinant Mouse VEGF proteins within the range of 15 6 pg ml 1000 pg ml FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 13 XIII CROSS REACTIVITY No detectable cross reactivity with other relevant proteins XIV REFERENCES 1 Basu S Nagy J A Pal S Vasile E Eckelhoefer A Bliss V S Manseau E J Dasgupta P S Dvorak F Mukhopadhyay D The neurotransmitter dopamine inhibits angiogenesis induced by vascular permeability factor vascular endothelial growth facto
3. 0 C Cell Lysates Collect cells and rinse cells with PBS Homogenize and lyse cells throughly in lysate solution Centrifuge cell lysates at approximately 10000 X g for 5 minutes to remove debris Aliquots of the cell lysates were removed and assayed Bone Tissue Extract demineralized bone samples in 4 M Guanidine HCl and protease inhibitors Dissolve the final sample in 2 M Guanidine HCl Tissue Homogenates Rinse tissue with PBS to remove excess blood chopped into 1 2 mm pieces and homogenize with a tissue homogenizer in PBS or in lysate solution lysate solution tissue net weight 10ml 1g i e Add 10ml lysate solution to 1g tissue Centrifuge at approximately 5000 X g for 5 minutes Assay immediately or aliquot and store homogenates at 20 C Avoid repeated freeze thaw cycles Urine Urinary samples should be cleared by centrifugation and then can be used directly without dilution Storage at 20 C 2 Mouse VEGF Standard Preparation Reconstitute the lyophilized Mouse VEGF Standard by adding 1 ml of Standard Sample Diluent to make the 10000 pg ml standard stock solution Allow FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES solution to sit at room temperature for 5 minutes then gently vortex to mix completely Use within one hour of reconstituting Two tubes of the standard 10 ng per tube are included in each kit Use one tube for each experiment Perform 2 fold serial dilutions of the top sta
4. E ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
5. Mouse VEGF ELISA Kit User Manual Catalog MBS825096 Sandwich Enzyme Linked Immunosorbent Assay for Quantitative Detection of Mouse VEGF Concentrations in Cell Culture Supernatants Serum Plasma Tissue Homogenates FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 1 l INTRODUCTION EE 2 ASSAY PRINCIPLES a iaae nsina oa aaa r Aana Le Yee 3 Ill KIT COMPONENTS sae aaeain E aa re retrace eter ES 4 IV STORAGEAND STABILITY runer ig A EE EA t ir entree 4 V MATERIALS REQUIRED BUT NOT PROVIDED eee eene 5 VI HEALTH AND SAFETY PRECAUTION S esee enne nennen nennen 5 VII REAGENT PREPARATION ei enenatis e Meere nennt 6 MIIL ASSAY PROCEDURE 2 erroe tine tne rer e enne tti nnb 9 IX ASSAY PROCEDURE 5 8 11 Xa IYRICALDATA ere n Ree 12 Xl SENSITIMUTY i uiii ere t Ne NI n rr i eene 12 XII SPECIEICITY Nee e et ERR aee rhe tei ee 12 XIII CROSS E nene tha tain nan 13 XIV REFERENCES con NONE eee aaa RA ARAS 13 XV TROUBLESHOOTING a a a 14 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES l INTRODUCTION Vascular permeability factor vascular endothelial growth factor V
6. PF VEGF a potent cytokine expressed by most malignant tumors has critical roles in vasculogenesis and both physiological and pathological angiogenesis VEGF produced by tumor cells potently stimulates endothelial cell proliferation and angiogenesis and plays a key role in the pathophysiology of several neoplasias may also play a pivotal role in mediating the development and progression of diabetic retinopathy VEGF a major regulator of angiogenesis binds to two receptor tyrosine kinases 1 and Fit 1 The VEGF gene is mapped by fluorescence in situ hybridization to chromosome 6p12 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Il ASSAY PRINCIPLES The Mouse VEGF ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of Mouse VEGF in Cell Culture Supernatants Serum Plasma Tissue Homogenates This assay employs an antibody specific for Mouse VEGF coated on a 96 well plate Standards and samples are pipetted into the wells and VEGF present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti Mouse VEGF antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted to the wells The wells are again washed a TMB substrate solution is added to the wells and color develops in proportion to the amount of VEGF bound The Stop S
7. ct Possible Cause e Insufficient washing Too much Streptavidin HRP Incubation time too long Development time too long Reagent added in incorrect order or incorrectly prepared e Standard has gone bad If there is a signal in the sample wells Assay was conducted from an incorrect starting point e Insufficient washing unbound Streptavidin HRP remaining Too much Streptavidin HRP Plate sealer or reservoir reused resulting in presence of residual Streptavidin HRP Plate not developed long enough Improper calculation of standard curve dilution Sample matrix is masking detection Samples contain protein levels above assay range Uneven temperature around work surface Solution Increase number of washes Increase time of soaking between in wash Check dilution titration Reduce incubation time Decrease the incubation time before the stop solution is added Review protocol e Check the condition of stored standard Reagents allows to come to 20 30 before performing assay Increase number of washes Carefully Check dilution Use fresh plate sealer and reagent reservoir for each step Increase substrate solution incubation time e Check dilution make new standard curve More diluted sample Recommended Dilute samples and run Again Avoid incubating plate in areas where environmental conditions vary Use plate sealer FOR RESEARCH US
8. ndards to make the standard curve within the range of this assay 15 6 pg ml 1000 pg ml as below Standard Sample Dilution Buffer serves as the zero standard 0 pg ml Standard Add Into 1000 pg ml 100 of the Standard 10000 pg ml 900 ul of the Standard Sample Diluent 500 pg ml 500 ul of the Standard 1000 pg ml 500 ul of the Standard Sample Diluent 250 pg ml 500 ul of the Standard 500 pg ml 125 pg ml 500 ul of the Standard 250 pg ml 62 5 pg ml 500 ul of the Standard 125 pg ml 31 25 pg ml 500 ul of the Standard 62 5 pg ml 15 625 pg ml 500 ul of the Standard 31 25 pg ml 0 ng ml 1 ml of the Standard Sample Diluent Note The standard solutions are best used within 2 hours The 10000 pg ml standard solution should be stored at 4 C for up to 12 hours or at 20 C for up to 48 hours Avoid repeated freeze thaw cycles 3 Biotin Labeled Detection Antibody Working Solution Preparation The Biotin Labeled Detection Antibody should be diluted in 1 100 with the Detection Antibody Diluent and mixed thoroughly The solution should be prepared no more than 2 hours prior to the experiment 4 Streptavidin HRP Working Solution Preparation The Streptavidin HRP should be diluted in 1 100 with the Streptavidin HRP Diluent and mixed thoroughly The solution should be prepared no more than 1 hour prior to the experiment FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 5 Wash Buffer
9. nized water 7 Computer and software for ELISA data analysis 8 Tubes to prepare standard or sample dilutions Vl HEALTH AND SAFETY PRECAUTIONS 1 Reagents provided in this kit may be harmful if ingested inhaled or absorbed through the skin Please carefully review the MSDS for each reagent before conducting the experiment 2 Stop Solution contains 2 N Sulfuric Acid H2SO and is an extremely corrosive agent Please wear proper eye hand and face protection when handling this material When the experiment is finished be sure to rinse the plate with copious amounts of running water to dilute the Stop Solution prior to disposing the plate FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Vil REAGENT PREPARATION 1 Sample Preparation Store samples to be assayed within 24 hours at 2 8 C For long term storage aliquot and freeze samples at 20 C Avoid repeated freeze thaw cycles Cell culture supernates Remove particulates by centrifugation assay immediately or aliquot and store samples at 20 C Serum Allow the serum to clot in a serum separator tube about 4 hours at room temperature Centrifuge at approximately 1000 X g for 15 minutes Analyze the serum immediately or aliquot and store samples at 20 C Plasma Collect plasma using heparin or EDTA as an anticoagulant Centrifuge for 15 minutes at 1500 X g within 30 minutes of collection Assay immediately or aliquot and store samples at 2
10. nto paper towels or other absorbent material 6 Add 100 ul of Streptavidin HRP Working Solution into each well and incubate the plate at 37 for 45 minutes 7 Wash plate 5 times with Wash Buffer Working Solution and each time let wash buffer stay in the wells for 1 2 minutes Discard the wash buffer and blot the plate onto paper towels or other absorbent material 8 Add 100 ul of TMB Substrate Solution into each well and incubate plate at 37 C in dark for 30 minutes 9 Add 100 ul of Stop Solution into each well The color changes into yellow immediately FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 10 Read the O D absorbance at 450nm in a microplate reader within 30 minutes after adding the Stop Solution For calculation the relative O D 450 the O D 450 of each well the O D 450 of Zero well The standard curve can be plotted as the relative O D 450 of each standard solution Y vs the respective concentration of the standard solution X The concentration of the samples can be interpolated from the standard curve Note If the samples measured were diluted multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES IX ASSAY PROCEDURE SUMMARY Prepare all reagents samples and standards Add 100 ul Standard or Sample Wash plate 3 times
11. olution changes the color from blue to yellow and the intensity of the color is measured at 450 nm FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES KIT COMPONENTS 96 well Plate Coated With Anti Mouse VEGF Antibody 12 x 8 Strips Mouse VEGF Standard 10 ngx2 Biotin Labeled Detection Antibody 100X 120 ul Streptavidin HRP 100X Standard Sample Diluent Detection Antibody Diluent Streptavidin HRP Diluent Wash Buffer 20X TMB Substrate Solution Stop Solution 12 ml Plate Adhesive Strips Technical Manual 1 Manual IV STORAGE AND STABILITY All kit components are stable at 2 to 8 C Standard recombinant protein should be stored at 20 C or 80 C recommended at 80 C after reconstitution Opened Microplate Wells or reagents may be store for up to 1 month at 2 to 8 C Return unused wells to the pouch containing desiccant pack reseal along entire edge Note the kit can be used within one year if the whole kit is stored at 20 C Avoid repeated freeze thaw cycles FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES V MATERIALS REQUIRED BUT NOT PROVIDED 1 Microplate reader capable of measuring absorbance at 450 nm 2 Adjustable pipettes and pipette tips to deliver 2 ul to 1 ml volumes 3 Adjustable 1 25 ml pipettes for reagent preparation 4 100 ml and 1 liter graduated cylinders 5 Absorbent paper 6 Distilled or deio
12. r Nature Med 7 569 574 2001 2 Poulaki V Mitsiades C S McMullan C Sykoutri D Fanourakis G Kotoula V Tseleni Balafouta S Koutras D A Mitsiades N Regulation of vascular endothelial growth factor expression by insulin like growth factor in thyroid carcinomas J Clin Endocr Metab 88 5392 5398 2003 3 Awata T Inoue K Kurihara S Ohkubo T Watanabe M Inukai K Inoue l Katayama S A common polymorphism in the 5 prime untranslated region of the VEGF gene is associated with diabetic retinopathy in type 2 diabetes Diabetes 51 1635 1639 2002 4 Soker S Takashima S Miao H Q Neufeld G Klagsbrun M Neuropilin 1 is expressed by endothelial and tumor cells as an isoform specific receptor for vascular endothelial growth factor Cell 92 735 745 1998 5 Wei M H Popescu C Lerman M I Merrill M J Zimonjic D B Localization of the human vascular endothelial growth factor gene VEGF at chromosome 6p12 Hum Genet 97 794 797 1996 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 14 XV TROUBLESHOOTING GUIDE Problem High signal and background in all wells No signal Too much signal whole plate turned uniformly blue Standard curve achieved but poor discrimination between point No signal when a signal is expected but standard curve looks fine Samples are reading too high but standard curve is fine Edge effe
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