Mouse TNF alpha ELISA Kit User Manual Catalog
Contents
1. NOT FOR OR THERAPEUTIC PROCEDURES XIII CROSS REACTIVITY No detectable cross reactivity with other relevant proteins XIV REFERENCES 1 Brenner D A O Hara M Angel P Chojkier M Karin M Prolonged activation of JUN and collagenase genes by tumour necrosis factor alpha Nature 337 661 663 1989 2 Nedwin G E Naylor S L Sakaguchi A Y Smith D Jarrett Nedwin J Pennica D Goeddel D V Gray P W Human lymphotoxin and tumor necrosis factor genes structure homology and chromosomal localization N cleic Acids Res 13 6361 6373 1985 3 Broudy V C Kaushansky K Segal G M Harlan J M Adamson J W Tumor necrosis factor type alpha stimulates human endothelial cells to produce granulocyte macrophage colony stimulating factor Proc Nat Acad Sci 83 7467 7471 1986 4 Wang A M A A Ladner M B Lin L S Strickler J Van Arsdell J N Yamamoto R Mark D F Molecular cloning of the complementary DNA for human tumor necrosis factor Science 228 149 154 1985 FOR RESEARCH USE ONLY NOT FOR eae 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 reading too high but stand2. plate Standards and samples are pipetted into the wells and TNF alpha present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti Mouse TNF alpha 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 TNF alpha 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 AD OR THERAPEUTIC PROCEDURES KIT COMPONENTS 96 well Plate Coated With Anti Mouse TNF alpha Antibody 12 x 8 Strips Mouse TNF alpha Standard 2ngx2 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 recommended at 80 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 who
3. 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 OR THERAPEUTIC PROCEDURES IX ASSAY PROCEDURE SUMMARY Prepare all reagents samples and standards Add 100 ul Standard or Sample Wash plate 3 times with Wash Buffer Working Solution Add 100 ul Biotin Labeled Detection Antibody Worki Wash plate 3 times with Wash Buffer Working dlution e v w Add 100 ul Streptavidin HRP Working Solution Wash plate 5 times wi h Wash Buffer Working Solution a NC Add 100 ul TMB Substrate Solution N lt N Add 100 ul Stop Solution A A 4 Read the plate at 450nm FOR RESEARCH USE ONLY NOT FOR eR 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 i 2 0 1 L 1 4 4 1 LJ 1 i 1 10 100 1000 Mouse TNF alpha Concentration pg ml XI SENSITIVITY The minimum detectable dose of Mouse TNF alpha is typically less than 4 pg ml XII SPECIFICITY The Mouse TNF alpha ELISA Kit allows for the detection and quantification of endogenous levels of natural and or recombinant Mouse TNF alpha proteins within the range of 15 625 pg ml 1000 pg ml FOR RESEARCH USE ONLY
4. 1 ml of Standard Sample Diluent to make the 2000 pg ml standard stock solution Allow FOR RESEARCH USE ONLY NOT FOR Ae 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 2 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 15 625 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 500 ul of the Standard 2000 pg ml 500 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 2000 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 a
5. Mouse TNF alpha ELISA Kit User Manual Catalog j Sandwich Enzyme Linked Immunosorbent Assay for Quantitative Detection of Mouse TNF alpha Concentrations in Cell Culture Supernatants Serum Plasma Tissue Homogenates For research use only Not for diagnostic or therapeutic procedures FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Ie INTRODUCTION EE 2 1 sabias 3 Ih KIT COMPONENT S p 4 IV STORAGE AND STABILITX ns ne kenne ne ein ener Lex ped tetas 4 V MATERIALS REQUIRED BUT NOT PROVIDED eee een 5 VI HEALTH AND SAFETY 5 enne nene nnne 5 VII REAGENT PREPARATION eene nennen enne eene 6 VIII ASSAY PROCEDURE e inert tnt or tp itr ee eene riter eren tecti 9 IX ASSAY PROCEDURE 0 enn tnmen enne nennen nene 11 KY PICA DIL 7L S po Ms Dp on IEEE 12 XI SENSITIVITY NM en e rere er erri eei nnn 12 XII SPECIFICITY iiiter ts Neu rer rere heredero Der iare 12 XIII CROSS REACTIVITY riscos Rum MUI soot deen eere entree 13 XIV REFERENCES IN mee RE a rera 13 XV TROUBLESHOOTING GUIDE eese nennen 14 FOR RESEARCH USE ONLY NOT FOR OR THERAPEUTIC PROCEDURES l INTRODUCTION
6. Tumor necrosis factor alpha TNF alpha or TNF is secreted by macrophages in response to inflammation infection and cancer Human Tumor Necrosis Factor TNF and Lymphotoxin TNF beta are cytotoxic proteins which have similar biological activities and share 3096 amino acid homology TNF alpha is produced by monocytes which can stimulate endothelial cells to produce the multilineage growth factor granulocyte macrophage colony stimulating factor and extend the role of this immunoregulatory protein to the regulation of hematopoiesis in vitro TNF is a soluble protein that causes damage to tumor cells but has no eff ct on normal cells Human TNF has been purified to apparent homogeneity as a 17 3 kilodalton protein from HL 60 leukemia cells and has showed cytotoxic and cytostatic activities against various human tumor cell lines The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids The mature protein begins at residue 77 leaving a long leader sequence of 76 amino acids TNF alpha has been mapped to human chromosome 6 2 FOR RESEARCH USE ONLY NOT FOR OR THERAPEUTIC PROCEDURES Il ASSAY PRINCIPLES The Mouse TNF alpha ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of Mouse TNF alpha in Cell Culture Supernatants Serum Plasma Tissue Homogenates This assay employs an antibody specific for Mouse TNF alpha coated on a 96 well
7. absorbent material Do NOT let the wells completely dry at any time 4 Add 100 ul of Biotin Labeled De tection 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 onto paper towels or other absorbent material 6 Add 100 yl 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 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
8. ard curve is fine Edge effect FOR RESEARCH USE ONLY NOT FOR US Possible Cause 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 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 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 incubating plate in areas where en
9. le 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 reviewthe 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 Q 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 a
10. liquot 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 theinal 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 TNF alpha Standard Preparation Reconstitute the lyophilized Mouse TNF alpha Standard by adding
11. nd 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 AD OR THERAPEUTIC PROCEDURES 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
12. vironmental conditions vary Use plate sealer OSTIC OR THERAPEUTIC PROCEDURES
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