User Manual
Contents
1. Centrifuge samples for 20 minutes at 1000xg to remove particulates Collect the supernatant for assaying Cell Lysates Collect and pellet the cells by centrifugation and remove the supernatant Wash the cells 3 times with PBS then resuspend in PBS Lyse the cells by ultrasonication 4 times Alternatively freeze the cells to 20 C and thaw to room temperature 3 times Centrifuge at 1500xg for 10 minutes at 2 8 C to remove cellular debris Collect the Supernatant for assaying Erythrocyte Lysates Centrifuge whole blood for 20 minutes at 1000xg to pellet the cells and remove the supernatant Wash the cells 3 times with PBS then resuspend in PBS Freeze 20 C thaw room temperature the cells 3 times Centrifuge at 5 000xg for 10 minutes at 2 8 C to remove cellular debris Collect the supernatant for assaying Erythrocyte lysates must be diluted with Sample Diluent before running Animal and Plant Tissue Supernatants Using a mortar and pestle grind the tissuesto a powder with liquid nitrogen Resuspend the powder in3x sample volume of sample extraction buffer 10 TCA and incubate overnight at 20 C Centrifuge at 8000rpm for 1h at 4 C to collect precipitated protein and decant the supernatant Add the same volume of ice cold 100 acetone centrifuge at 8000rpm for 15min at 4 C then dry vacuum deposition in reserve Add lysis buffer 2 7g urea 0 2g CHAPS add dH20 to 5ml incubate at room temperature for 30 minutes then2. Prior to running valuable samples LSBio recommends that the user run a preliminary experiment using the supplied controls in order to validate the assay To minimize external influence on the assay performance operational procedures and lab conditions such as room temperature humidity and incubator temperature should be strictly controlled It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end The kit should not be used beyond the expiration date on the kit label 14 Prepare all reagents samples and standards Add 100 ul standard or sample to each well and incubate for 2 5 hours at room temperature or overnight at 4 C Aspirate and wash 4 times Add 100 ul prepared Detection Antibody and incubate for 1 hour at room temperature Aspirate and wash 4 times Add 100 ul prepared HRP Streptavidin Solution and incubate for 45 minutes at room temperature Aspirate and wash 4 times Add 100 ul Substrate Solution and incubate for 30 minutes at 37 C Add 50 ul Stop Solution Read immediately at 450nm gt ON gt lt D JJ O O m J C JJ m UN GC lt UI CALCULATION OF RESULTS Average the duplicate readings for each standard control and sample and subtract the average zero standard optical density Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic 4 PL cu
3. overnight at 4 C with gentle shaking 2 Aspirate the liquid from each well and wash 4 times Wash by adding approximately 300 ul of Wash Buffer using asquirt bottle multi channel pipette manifold dispenser or automated washer Allow each wash to sit for 1 2 minutes before completely aspirating After the last wash aspirate to remove any remaining Wash Buffer then invert the plate and tap against clean absorbent paper 3 Add 100 ul of Detection Antibody Working Solution to each well and gently agitate to ensure thorough mixing Incubate for 1 hour at room temperature 4 Aspirate and wash the wells as outlined in step 2 5 Add 100 ul of HRP Streptavidin Working Solution to each well and incubate for 45 minutes at room temperature with gentle shaking 6 Aspirate and wash the wells as outlined in step 2 7 Add 100 ul of Substrate Solution to each well and incubate for 30 minutes at room temperature in the dark with gentle shaking Monitor periodically until optimal color development has been achieved 8 Add 50 ul of Stop Solution to each well and record the total development time The blue color will change to yellow If color change does not appear uniform gently tap the plate to ensure thorough mixing The Stop Solution should be added to wells in the same order and timing as the substrate solution 9 Determine the optical density OD value of each well immediately using a microplate reader set to 450 nm 12 ASSAY P
4. by chemical lysis buffers may result in inaccurate results Due to the factors including cell viability cell number or sampling time samples from cell culture supernatant may not be detected by the kit Samples should be brought to room temperature 18 25 C before performing the assay without the use of extra heating Sample concentrations should be predicted before being used In the assay If the sample concentration is not within the range of the standard curve users must determine the optimal sample dilutions for their particularexperiments LSBio is responsible for the quality and performance of the kit components but is NOT responsible for the performance of customer supplied samples used with the kit REAGENT PREPARATION Bring all reagents to room temperature 18 25 C before use 14 Assay Diluent Prepare 75 ml of 1x Assay Diluent by diluting the supplied 15 ml of 5x Assay Diluent concentrate with 60 ml of deionized or distilled water Assay Diluent can be stored at 4 C once prepared 1x Wash Buffer If crystals have formed in the concentrate warm to room temperature and mix it gently until crystals have completely dissolved Prepare 400 ml of Working Wash Buffer by diluting the supplied 20 ml of 20x Wash Buffer Concentrate with 380 ml of deionized or distilled water Wash Buffer can be stored at 4 C once prepared Detection Antibody Concentrate and Working Solution Briefly spin down the Detection Antibody and add 100
5. D266 FGF inducible 14 FN14 Tweak receptor TWEAKR Specificity Recombinant and natural Mouse TNFRSF12A FN14 Add cross reactivity data Sample Types This kit is recommended for use with M ouse serum plasma and tissue homogenates Use with other samplertypes Is not supported Detection Range To be determined Sensitivity Lower Limit of Detection To be determined Performance Characteristics InterAssay CV lt To be determined IntraAssay CV lt To be determined INTENDED U SE This Sandwich ELISA Enzyme Linked Immunosorbent Assay kit is intended for the in vitro guantitative determination of M ouse TNFRSF12A FN14 concentrations in serum plasma and tissue homogenates Use with other sample types is not supported ASSAY PRINCIPLE This assay is based on the sandwich ELISA principle Each well of the supplied microtiter plate has been pre coated with a target specific capture antibody Standards or samples are added to the wells and the target antigen binds to the capture antibody Unbound Standard or sample is washed away A biotin conjugated detection antibody is then added which binds to the captured antigen Unbound detection antibody is washed away An Avidin Horseradish Peroxidase HRP conjugate is then added which binds to the biotin Unbound Avidin HRP conjugate is washed away A TMB substrate is then added which reacts with the HRP enzyme resulting in color development A sulfuric acid stop solution is a
6. DARD PREPARATION The following are instructions for the preparation of a Standard dilution series which will be used to generate the standard curve The standard curve is then used to determine the concentration of target antigen in unknown samples see the Calculation of Results section The following will prepare sufficient volumes to run the Standard dilution series in duplicate Standard dilutions should be used immediately once prepared Standard Stock Solution x Units Reconstitute 1 tube of lyophilized Standard with x ml of Sample Diluent Incubate at room temperature for 10 minutes with gentle agitation avoid foaming D1 x Units Pipette a ul of Stock Standard into X ul of Sample Diluent D2 x Units Pipette 250ul of DL into 250ul of Sample Diluent D3 x Units Pipette 250ul of D2 into 250ul of Sample Diluent D4 x Units Pipette 250ul of D3 into 250ul of Sample Diluent D5 x Units Pipette 250ul of D4 into 250ul of Sample Diluent D6 x Units Pipette 250ul of D5 into 250ul of Sample Diluent D7 x Units Pipette 250ul of D6 into 250ul of Sample Diluent Zero Standard 0 Units Use Sample Diluent alone CVUNONONONON Wee e bee Standard D1 D7 Zero Stock 10 REAGENT PREPARATION NOTES 1 2 10 11 12 It is highly recommended that standard curves and samples are run in duplicate within each experiment Once resuspended standard should be used immediately or placed in long term storage a
7. LifeSpan BioSciences Inc BioSciences Inc Sample Mouse TNFRSF12A FN14 Sandwich ELISA Kit User M anual Catalog No LS F301 Please read this manual carefully before starting your experiment This kit is for Research Use Only Not for Diagnostic Use This kit is not approved for use in humans or for clinical diagnosis Assay Specifications waarnemen nnen nennen 1 LENG sie U E asa aa ag an ag aaa Ka Aa A tr ga aa ga aaa ag 2 ASSav PTINCIDI C sana aa aaa agan aa a a Laa aa nana An 2 Assay Principle IMaQ ccsseseeerere ay 3 Kit COMPO MEMES annen ed Nr eens 4 A 0 em ee enn 4 Other Required Supplies o a Aarssen 4 Sample Collectlon 0r regan isene Ma eneren errereen 5 Sample Collection Notes Aas ska nara aae aranana aranana nan 7 Reagent Preparation nt inie an anaa aaa nan anna 6 Sample Preparation ik P W rasana na nana anana anana nee 9 Standard Preparation nonnen enneeen ennen 10 Reagent Preparation Notes ennnnnern ennen 11 ASSAY PLQCEAUPENge en eerernenereeneneerersaneveersneerevenn 12 Assay Procedure Notes sooner aa nen nan anan 13 Assay Procedure Summary anneer eenn 15 Calculation of RESUIES users sarnana renane aana ngaen naa 16 Troubleshooting Guide anas anana ennnnen een 17 Troubleshooting Guide continued 18 ASSAY SPECIFICATIONS Target TNFRSF12A FN14 Synonyms TNFRSF12A FN14 TNFRSF12A tumor necrosis factor receptor superfamily member 12A CD266 antigen C
8. On disposal flush with large volumes of water to prevent accumulation Returns Refunds Cancelations Any problems with LifeSpan products must be reported to LifeSpan within 10 days of product receipt The customer must obtain written authorization from LifeSpan before returning items To request that goods be returned please contact LifeSpan Technical Support If an error by LifeSpan BioSciences results in shipment of an incorrect order LifeSpan will at its option either ship a replacement order at no charge or credit the customer s account for the original product shipped in error Returns and cancelations may be subject to a 30 restocking fee Conditions amp Warranty All LifeSpan products are intended for Research Use Only and are not for use in Human therapeutic or diagnostic applications The information supplied with each product is believed to be accurate but no warranty or guarantee is offered for the products because the ultimate conditions of use are beyond LifeSpan s control The information supplied with each product is not to be construed as a recommendation to use this product in violation of any patent and LifeSpan will not be held responsible for any infringement or other violation that may occur with the use of its products Under no event will LifeSpan be responsible for any loss of profit or indirect consequential damage including but not limited to personal injuries resulting from use of these products LifeSpan s liabil
9. ROCEDURE NOTES 1 ELISA Plate Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate Removed strips should be placed in a sealed bag containing desiccant and stored at 4 C Solutions To avoid cross contamination change pipette tips between additions of each standard between sample additions and between reagent additions Also use separate reservoirs for each reagent Applying Solutions All solutions should be added to the bottom of the ELISA plate well Avoid touching the inside wall of the well Avoid foaming when possible Assay Timing The interval between adding sample to the first and last wells should be minimized Delays will increase the incubation time differential between wells which will significantly affect the experimental accuracy and repeatability For each step in the procedure total dispensing time for addition of reagents or samples should not exceed 10 minutes Incubation To prevent evaporation and ensure accurate results proper adhesion of plate sealers during incubation steps Is necessary Do not allow wells to sit uncovered for extended periods of time between incubation steps Do not let wells dry out at any time during the assay Strictly observe the recommended incubation times and temperatures Washing Proper washing procedure is critical Insufficient washing will result in poor precision and falsely elevated absorbance readings Residual liquid in the react
10. centrifuge at 8000rpm for 15min at 4 C Collect the Supernatant for assaying Plasma Collect plasma using EDTA or heparin as an anticoagulant Centrifuge samples for 15 minutes at 1000xg at 2 8 C within 30 minutes of collection Collect the supernatant for assaying Platelet Poor Plasma Collect plasma using EDTA as an anticoagulant Centrifuge samples for 15 minutes at 1000xg at 2 8 C within 30 minutes of collection It is recommended that samples should be centrifuged for 5 10 minutes at 10 000xg for complete platelet removal Collect the supernatant for assaying Sperm and Seminal Plasma Allow semen to liquefy at room temperature or 37 C After liquefaction centrifuge at 2 000xg for 10 15 minutes Collect seminal plasma supernatant for assaying Wash the precipitated protein 3 times with PBS then resuspend in PBS Lyse the cells by ultrasonication then centrifuge at 2 000xg for 10 15 minutes Collect the supernatant for assaying Serum Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at 4 C before centrifugation for 20 minutes at approximately 1000xg Collect the supernatant for assaying Tissue Homogenates Because preparation methods for tissue homogenates vary depending upon tissue type users should research tissue specific conditions independently The following is one example only Rinse tissues in PBS to remove excess blood and weigh before homogenization Fi
11. dded to terminate color development reaction and then the optical density OD of the well is measured at a wavelength of 450 nm 2 nm The OD of an unknown sample can then be compared to an OD standard curve generated using known antigen concentrations in order to determine its antigen concentration AK de A AN mm DS B B Apoquuy uoaa payevugorg xardwo5 dH4H upmedans den uabewous BNL AA ANL JOY JIdONldd AVSSY KIT COMPONENTS PlateSealers TN KIT STORAGE The unopened kit may be stored for up to 6 months at 2 8 C or 1 year at 20 C Once reconstituted Standard should be stored at 20 C 80 C Avoid freeze thaw cycles Once opened all other reagents can be stored up to 1 month at 2 8 C Once individual reagents are opened it is recommended that the kit be used within 1 month Unused Strip Plate wells should be stored at 2 8 C in a sealed bag containing desiccant in order to minimize exposure to moisture Do not use the kit beyond its expiration date OTHER REQUIRED SUPPLIES Microplate reader with 450nm wavelength filter High precision pipette and sterile pipette tips Eppendorf tubes 37 C incubator Deionized or distilled water Absorbent paper SAM PLE COLLECTION This assay is recommended for use with Human in serum plasma and tissue homogenates Use with other sample types is not supported The sample collection protocols below have been provided for your reference Breast Milk
12. ion wells should be patted dry against absorbent paper during the washing process Do not put absorbent paper directly into the reaction wells Controlling Substrate Reaction Time After the addition of the TMB Substrate periodically monitor the color development Stop color development before the color becomes too deep by adding Stop Solution Excessively strong color will result in inaccurate absorbance reading Reading The microplate reader should be preheated and programmed prior to use Prior to taking OD readings remove any residual liquid or fingerprints from the underside of the plate and confirm that there are no bubbles in the wells 13 10 11 12 13 14 15 16 Reaction Time Control Control reaction time should be strictly followed as outlined Stop Solution The Stop Solution contains an acid therefore proper precautions should be taken during its use such as protection of the eyes hands face and clothing M iking During incubation times the use of a micro oscillator at low frequency is recommended Sufficient and gentle mixing is particularly important in producing reliable results Kits from different batches may be a little different in detection range sensitivity and color developing time Please perform the experiment exactly according to the supplied instructions Due to inter and intra assay variability it is recommended that appropriate carry over controls be included between assays
13. ity toany user of Products for damages that do not result from any fault of the user will be limited to replacement of the Product s only and in no event shall LifeSpan s liability exceed the actual price received by LifeSpan for the Product s at issue LifeSpan shall not be liable for any indirect special incidental or consequential damages LIFESPAN FURTHER DISCLAIMS ANY AND ALL EXPRESS AND IMPLIED OR STATUTORY WARRANTIES WITH RESPECT TO THE PRODUCTS INCLUDING BUT NOT LIMITED TO ANY IM PLIED WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE LifeSpan disclaims any and all responsibility for any injury or damage which may be caused by the fault of the user For Research Use Only Not approved for use in Rats or for clinical diagnosis Be LSBio LifeSpan BioSciences Inc 2401 Fourth Avenue Suite 900 Seattle WA 98121 Tel 206 464 1554 Fax Toll Free North America 866 206 6909 Fax International Or Local 206 577 4565 Technical Support LSBio com 19
14. nd volumes ensure correct preparation Deep color but low Plate reader settings Verify the wavelength value not optimal and filter setting in the plate reader Open the Plate Reader ahead to pre heat 17 TROUBLESHOOTING GUIDE CONTINUED Large CV Inaccurate pipetting Check pipettes High background Concentration of Use recommended detector too high dilution factor Plate is insufficiently Review the manual washed for proper washing Instructions If using a plate washer check that all ports are unobstructed Contaminated wash M ake fresh wash buffer buffer Low sensitivity Improper storage of All the reagents the ELISA kit should be stored according to the instructions Stop solution not Stop solution should added be added to each well before measurement 18 Important Note During shipment small volumes of product will occasionally become entrapped in the seal of the product vial We recommend briefly centrifuging the vial to dislodge any liquid in the container s cap prior to opening Warning This reagent may contain sodium azide and sulfuric acid The chemical physical and toxicological properties of these materials have not been thoroughly investigated Standard Laboratory Practices should be followed Avoid skin and eye contact inhalation and ingestion Sodium azide forms hydrazoic acid under acidic conditions and may react with lead or copper plumbing to form highly explosive metal azides
15. nely mince tissues and homogenize them in 5 10mL of PBS with a glass homogenizer once Lyse the cells by ultrasonication or freeze 20 C thaw room temperature 3 times Centrifuge homogenate at 5000xg for 5 minutes Collect the Supernatant for assaying Urine Aseptically collect the first urine of the day mid stream voided directly into a sterile container Centrifuge to remove particulate matter and collect the supernatant for assaying Cell culture supernatants cerebrospinal follicular and lung lavage fluids saliva sweat tears and other biological fluids Centrifuge samples for 20 minutes at 1000xg to remove particulates Collect the Supernatant for assaying 1xPBS 0 02mol L pH7 0 7 2 SAMPLE COLLECTION NOTES 1 LSBio recommends that samples are used immediately upon preparation Alternatively samples stored at 2 8 C should be used within 5 days For long term storage sample aliquots should be prepared and stored at 20 C if used within 1 month or 80 C If used within 6 months Long term storage can result in protein degradation and denaturation which may result in inaccurate results Avoid repeated freeze thaw cycles for all samples In the event that a sample type not listed above is intended to be used with the kit it is recommended that the customer conduct validation experiments in order to be confident inthe results Due to chemical interference the use of tissue or cell extraction Samples prepared
16. rve fit As an alternative construct a standard curve by plotting the mean absorbance for each standard on the x axis against the concentration on the y axis and draw a best fit curve through the points on the graph The data may be linearized by plotting the log of the target antigen concentrations versus the log of the O D and the best fit line can be determined by regression analysis Use of a commercial software program such as CurveExpert is recommended for performing this calculation This procedure will produce an adequate but less precise fit of the data If samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor Typical Data The following standard curve Is an example only and should not be used to calculate results for tested samples A new standard curve must be generated for each set of samples tested Standard Curve Image Concentration Units Optical Density 16 TROUBLESHOOTING GUIDE Poor standard curve Inaccurate pipetting Check pipettes Improper standard Briefly spin the vial of dilution standard and dissolve the powder thoroughly by a gentle mix Wells not completely Completely aspirate aspirated wells between steps Low signal Too brief incubation Ensure sufficient times incubation time Incorrect assay Use recommended temperature Incubation temperature Bring substrate to room temperature before use Inadequate reagent Check pipettes a
17. t 20 80 C In order to avoid repeat freeze thaws prepare one time use aliquots of standard for long term storage at 20 80 C All solutions prepared from concentrates are intended for one time use Do not reuse solutions Do not prepare Standard dilutions directly in wells Prepared Reagents may adhere to the tube wallor cap during transport centrifuge tubes briefly before opening All solutions should be gently mixed prior to use Reconstitute stock reagents in strict accordance with the instructions provided To minimize imprecision caused by pipetting ensure that pipettes are calibrated Pipetting volumes of lessthan 10uL is not recommended Substrate Solution is easily contaminated sterility precautions should be taken Substrate Solution should also be protected from light Do not substitute reagents from one kit lot to another Use only those reagents supplied within this kit Due to the antigen specificity of the antibodies used in this assay native or recombinant proteins from other manufacturers may not be detected by this kit 11 ASSAY PROCEDURE Bring all reagents and samples to room temperature without additional heating and mix thoroughly by gently swirling before pipetting avoid foaming Prepare all reagents working standards and samples as directed in the previous sections 1 Add 100 ul of Standard Blank or Sample per well cover with a plate sealer and incubate for 2 5 hours at room temperature or
18. ul of 4x Assay Diluent to prepare the Detection Antibody Concentrate The Detection Antibody Concentrate can be stores at 4 C for 5 days Calculate the volume of Detection Antibody Working Solution needed for your particular experiment and prepare that volume by diluting the Detection Antibody Concentrate 80 fold 1 80 with 1x Assay Diluent HRP Streptavidin Working Solution Gently mix the stock solution before use Calculate the volume of HRP Streptavidin Working Solution needed for your particular experiment and prepare that volume by diluting the HRP Streptavidin Conjugate Solution 800 fold 1 800 with 1x Assay Diluent Prepared HRP Streptavidin working solution must be prepared fresh for each experiment and cannot be stored TMB Substrate Solution Using sterile techniques remove the needed volume of TMB Substrate Solution for the number of wells you are planning to run Dispose of unused TMB Substrate Solution rather than returning it to the stock container SAM PLE PREPARATION Please predict the concentration of your samples before assaying The resulting optical density OD values of your sample must fall within the OD readings of the standard curve in order for the calculated antigen concentration to be accurate A dilution series of each sample may be necessary Running duplicate wells for each sample is recommended All samples should be diluted with 1x Assay Diluent List of any specific sample preparation details STAN
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