Human TGF beta 1 ELISA Kit User Manual Catalog
Contents
1. RESEARCH USE ONLY NOT FOR 0 OR THERAPEUTIC PROCEDURES I INTRODUCTION Transforming growth factor beta 1 TGF beta 1 is a multifunctional peptide that controls proliferation differentiation and other functions in many cell types Many cells synthesize TGF beta and essentially all of them have specific receptors for this peptide TGF beta regulates the actions of many other peptide growth factors and determines a positive or negative direction of their effects TGF beta 1 is known for its potent and diverse biological effects including immune regulation and cell growth and differentiation TGF beta 1 is also an important mediator of bone remodeling TGF beta 1 a potent keratinocyte growth inhibitor has been shown to be overexpressed in keratinocytes in certain inflammatory skin diseases and has been thought to counteract the effects of other growth factors at the site of inflammation TGF beta 1 a multifunctional cytokine with fibrogenic properties has been implicated in the pathogenesis of the vascular and target organ complications of hypertension TGF beta 1 may also regulate blood pressure via stimulation of endothelin 1 and or renin secretion TGF beta 1 is secreted as a latent form which consists of its mature form and a latency associated peptide beta 1 LAP in either the presence or the absence of additional latent TGF beta 1 binding protein FOR RESEARCH USE ONLY NOT FOR USE I NOSTIC OR THERAPEUTIC PROCEDURES Il ASSAY PRIN2. CIPLES The Human TGF beta 1 ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of Human TGF beta 1 in Cell Culture Supernatants Serum Plasma Urine Tissue Homogenates This assay employs an antibody specific for Human TGF beta 1 coated on a 96 well plate Standards and samples are pipetted into the wells and TGF beta 1 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti Human TGF beta 1 antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted to the wells wells are again washed a TMB substrate solution is added to the wells and color develops in proportion to the amount of TGF beta 1 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm FOR RESEARCH USE ONLY NOT FOR 0 OR THERAPEUTIC PROCEDURES KIT COMPONENTS Human TGF beta 1 Standard 4ngx2 Biotin Labeled Detection Antibody 100X 120 ul Streptavidin HRP 100X Activation Solution A Activation Solution B Standard Sample Diluent Detection Antibody Diluent Streptavidin HRP Diluent Wash Buffer 20X 30 ml TMB Substrate Solution Stop Solution 12 ml Plate Adhesive Strips 3 Strips Technical Manual IV STORAGE AND STABILITY All kit components are stable at 2 to 8 C Standard recombinant prote
3. Human TGF beta 1 ELISA Kit User Manual Catalog MBS824677 Sandwich Enzyme Linked Immunosorbent Assay for Quantitative Detection of Human TGF beta 1 Concentrations in Cell Culture Supernatants Serum Plasma Tissue Homogenates Urine For research use only Not for diagnostic or therapeutic procedures FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Ie EE 2 1 ASSAY PRINCIPLES 3 Ih KIT GOMPONENT S p 4 IV STORAGE AND STABILITY eren tetas 4 V MATERIALS REQUIRED BUT NOT nnn 5 VI HEALTH AND SAFETY PRECAUTIONS isisisi atinaeina 5 VII REAGENT 44 ile Seen 6 ASSAY PROCEDU RE 2 neon enr rr t titre 9 IX ASSAY PROCEDURE 5 11 XD YPICAL ia MM o cocotte 12 SENSITIVITY e N e rere nnn 12 XII SPECIFICITY rete eres 12 XIII CROSS REACTIVITY 1 fierent eite 13 XIV REFERENCES ia 13 XV TROUBLESHOOTING GUIDE seen een nnne nnne nenne 14 XVI TECHNICAL SUPPOM INK inerenti teen et enata 15 XVII ej Sem c cem EET 15 FOR
4. IC 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 20 C Cell Lysates Collect cells and rinse cells with PBS Homogenize and lyse cells throughly in lysate solution Centrifuge celllysates 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
5. 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 Activate the sample FOR RESEARCH USE ONLY NOT FOR 0 OR THERAPEUTIC PROCEDURES Cell culture supernate urine add activating reagent pro rata i e add 20 ul of Activation Solution A into 100 ul of sample 10 minutes later add 20 ul of Activation Solution B PH 7 0 7 6 Serum plasma add activating reagent pro rata i e add 20 ul of Activation Solution A into 40 ul of sample 10 minutes later add 20 ul of Activation Solution B PH 7 0 7 6 It is unnecessary to activate the recombinant TGF beta 1 Note Sample was diluted partly after adding activating reagent so please pay attention to this when calculate target protein concentration 2 Human TGF beta 1 Standard Preparation Reconstitute the lyophilized Human TGF beta 1 Standard by adding 1 ml of Standard Sample Diluent to make the 4000 pg ml standard stock solution Allow 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 4 ng per tube are included in each kit Use one tube for each experiment Perform 2 fold serial dilutions of the top standards to make the standard curve within the range of this assay 31 25 pg ml 2000 pg ml as below Standard Sample D
6. ating plate in areas where environmental conditions vary Use plate sealer NOSTIC OR THERAPEUTIC PROCEDURES XVI TECHNICAL SUPPORT For troubleshooting information or assistance please go online XVII NOTES FOR RESEARCH USE ONLY NOT FOR USE I NOSTIC OR THERAPEUTIC PROCEDURES
7. ilution Buffer serves as the zero standard 0 pg ml Standard 2000 pg ml 1000 pg ml 500 pg ml 250 pg ml 125 pg ml 62 5 pg ml 31 25 pg ml 0 ng ml FOR RESEARCH USE ONLY NOT FOR USE I Add 500 ul of the Standard 4000 pg ml 500 ul of the Standard 2000 pg ml 500 ul of the Standard 1000 pg ml 500 ul of the Standard 500 pg ml 500 ul of the Standard 250 pg ml 500 ul of the Standard 125 pg ml 500 ul of the Standard 62 5 pg ml 1 ml of the Standard Sample Diluent Into 500 ul of the Standard Sample Diluent 500 ul of the Standard Sample Diluent 500 ul of the Standard Sample Diluent 500 ul of the Standard Sample Diluent 500 ul of the Standard Sample Diluent 500 ul of the Standard Sample Diluent 500 ul of the Standard Sample Diluent NOSTIC OR THERAPEUTIC PROCEDURES Note The standard solutions are best used within 2 hours The 4000 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 sh
8. in 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 Q 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 deionized 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 Sulfuric Acid gt 504 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 0 OR THERAPEUT
9. l Standard or Sample Wash plate 3 times with Wash Buffer Working Solution Wash plate 3 times with Wash Buffer Working Solution m v hd Add 100 ul Streptavidin HRP Wa g Solution b Wash plate 5 times Wash Buffer Working Solution Add 100 ul TN y ubstrate Solution to Add 100 ul Stop Solution A Readthe plate at 450nm FOR RESEARCH USE ONLY NOT FOR Q OR THERAPEUTIC PROCEDURES X TYPICAL DATA The standard curve is for demonstration only A standard curve must be run with each assay 10 oa Absorbance 450nm 0 1 1 1 L 11144 i 1 2 ee ee L 1 1 a See 10 100 1000 10000 Human TGF beta 1 Concentration pg ml SENSITIVITY The minimum detectable dose of Human TGF beta 1 is typically less than 7 pg ml SPECIFICITY The Human TGF beta 1 ELISA Kit allows for the detection and quantification of endogenous levels of natural and or recombinant Human TGF beta 1 proteins within the range of 31 25 pg ml 2000 pg ml FOR RESEARCH USE ONLY NOT FOR OR THERAPEUTIC PROCEDURES XIII CROSS REACTIVITY No detectable cross reactivity with other relevant proteins XIV REFERENCES 1 Sporn M B Roberts A B Wakefield L M Assoian R K Transforming growth factor beta biological function and chemical structure Science 233 532 534 1986 2 Blanchette F Day R Dong W Laprise M H Dubois C M TGF beta 1 regulates gene ex
10. ould be prepared no more than 1 hour prior to the experiment 5 Wash Buffer 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 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 minu
11. pression of its own converting enzyme furin J Clin Invest 99 1974 1983 1997 3 Janssens K Gershoni Baruch R Guanabens N Migone N Ralston S Bonduelle M Lissens W Van Maldergem L Vanhoenacker F Verbruggen L Van Hul W Mutations in the gene encoding the latency associated peptide of TGF beta 1 cause Camurati Engelmann disease Nature Genet 26 273 275 2000 4 Li A G Wang D Feng X H Wang X J Latent TGF beta 1 overexpression in keratinocytes results in a severe psoriasis like skin disorder EMBO J 23 1770 1781 2004 5 Li B Sharma V Singh T Suthanthiran M August P TGF beta 1 DNA polymorphisms protein levels and blood pressure Hypertension 33 271 275 1999 6 Saito T Kinoshita A Yoshiura K Makita Y Wakui K Honke K Niikawa N Taniguchi N Domain specific mutations of a transforming growth factor TGF beta 1 latency associated peptide cause Camurati Engelmann disease because of the formation of a constitutively active form of TGF beta 1 J Biol Chem 276 11469 11472 2001 FOR RESEARCH USE ONLY NOT FOR O OR THERAPEUTIC PROCEDURES 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
12. reading too high but standard curve is fine Edge effect FOR RESEARCH USE ONLY NOT FOR USE I 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 Standard has gone bad If there is a signal in the sample wells Assay was conducted from an incorrect starting point Insufficient washing unbound Streptavidin HRP remaining Too much Streptavidin HRP e 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 e 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 Check the condition of stored standard Reagents allows to come to 20 30 C 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 Check dilution make new standard curve More diluted sample Recommended Dilute samples and run Again Avoid incub
13. tes Discard the Wash Buffer Working Solution and blot the plate onto paper towels or other absorbent material 6 Add 100 ul of Streptavidin HRP Working Solution into each well and incubate the plate at 37 C 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 0 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 I NOSTIC OR THERAPEUTIC PROCEDURES IX ASSAY PROCEDURE SUMMARY Prepare all reagents samples and standards Add 100 u
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