User Manual - MyBioSource
Contents
1. 20 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 4 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES EXPERIM ENTAL LAYOUT The following is an example of how to layout a study A dilution series of the positive control Standard should be run in duplicate or triplicate with the last well in each series being the negative control blank Samples should also be run in duplicate or triplicate Unknown samples should be run as a dilution series in order to identify the optimal dilution that produces an OD reading within the OD range of the positive control Standard dilution series Example 1 Standard Curve and dilution series of an unknown sample 1 2 3 4 A Standard Dilution 1 Standard Dilution 1 Sample Sample B Standard Dilution 2 Standard Dilution 2 C Standard Dilution 3 Standard Dilution 3 D Standard Dilution 4 Standard Dilution 4 E Standard Dilution 5 Standard Dilution 5 Sample Sample 1 10k 1 10k re AD A Sample Sample F Standard Dilution 6 Standard Dilution 6 1 100k 1 100k ae ra Sample Sample G Standard Dilution 7 Standard Dilution 7 1 1 002. don t wash 3 Add 100ul of Detection Reagent A working solution to each well cover with a plate sealer and gently agitate to ensure thorough mixing Incubate for 1 hour at 37 C 4 Aspirate the liquid from each well and wash 3 times Wash by adding approximately 350 ul of Wash Buffer using a squirt 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 4 Add 100ul of Detection Reagent B working solution to each well cover with a new plate sealer and incubate for 1 hour at 37 C 5 Aspirate the liquid from each well and wash 5 times as outlined in step 4 6 Add 90ul of Substrate Solution to each well cover with a new plate sealer and incubate for 15 30 minutes at 37 C Protect from light and monitor periodically until optimal color development has been achieved 7 Add 50ul of Stop Solution to each well The blue color will change to yellow immediately 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 8 Determine the optical density OD value of each well immediately using a microplate reader set to 450 nm 13 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC
3. Cathepsin S Cat s CATS Specificity Recombinant and natural Mouse CTSS Cathepsin S No significant cross reactivity or interference between CTSS Cathepsin S and analogs was observed This claim is limited by existing techniques therefore cross reactivity may exist with untested analogs Sample Types This kit is recommended for use with Mouse Plasma Serum and Tissue Homogenates Use with other sample types is not supported Detection Range 78 13 5000 pg ml Sensitivity Typically less than 25 2 pg ml Performance Intra Assay CV lt 10 Inter Assay CV lt 12 Limitations This kit is for Research Use Only and is not intended for diagnostic use This kit is not approved for use in humans or for clinical diagnosis 1 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 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
4. PROCEDURES ASSAY PROCEDURE 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 2 Solutions In the event that Detection Reagent A working solution appears cloudy warm to room temperature and mix gently until solution appears uniform 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 3 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 4 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 5 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 temperatu
5. products because the ultimate conditions of use are beyond our 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 we will not be held responsible for any infringement or other violation that may occur with the use of its products Under no event will we be responsible for any loss of profit or indirect consequential damage including but not limited to personal injuries resulting from use of these products Our liability to any 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 our liability exceed the actual price received by us for the Product s at issue We shall not be liable for any indirect special incidental or consequential damages WE FURTHER DISCLAIMS ANY AND ALL EXPRESS AND IMPLIED OR STATUTORY WARRANTIES WITH RESPECT TO THE PRODUCTS INCLUDING BUT NOT LIMITED TO ANY IMPLIED WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE We disclaim 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 humans or for clinical diagnosis 20 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
6. 0k 1 1 000k Sample Sample H Negative Control Negative Control 1 10 000k 1 10 000k Example 2 Standard Curve and samples run in duplicate 1 2 3 4 A Standard Dilution 1 Standard Dilution 1 Sample A Sample E B Standard Dilution 2 Standard Dilution 2 Sample A Sample E C Standard Dilution 3 Standard Dilution 3 Sample B Sample F D Standard Dilution 4 Standard Dilution 4 Sample B Sample F E Standard Dilution 5 Standard Dilution 5 Sample C Sample G F Standard Dilution 6 Standard Dilution 6 Sample C Sample G G Standard Dilution 7 Standard Dilution 7 Sample D Sample H H Negative Control Negative Control Sample D Sample H 5 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES SAM PLE COLLECTION This assay is recommended for use with Mouse Plasma Serum and Tissue Homogenates Use with other sample types is not supported The sample collection protocols below have been provided for your reference Breast Milk 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 supernat
7. Mouse CTSS Cathepsin S ELISA Kit Sandwich ELISA User Manual Catalog No MBS249228 It is important that you read this entire 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 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Assay Spe CifiCatiOnS ccccccccceccceceeceeeeeeeeeseeeseeseesseeeseeees 1 ASSAY Principles ee dig 2 Kit COMPONENTS SE EE SE SEE RE EG NG EE Eg Ge EE Gee Ee ge 4 KILSLORAE SE DE RR EDE MT eres 4 Other Required Supplies sesse ee ee 4 Experimental LayOut cccccccccccccccececeececeeeeseeseeeeeeseeeees 5 Sample Collection EE ER ER EE ER ee ee ee ee ee 6 Sample Collection Note Ee Ee 8 Sample PreBaratio ss EE N Ge docdseas cave ia 9 Standard Preparation f hef ss EE EG Ee 10 Reagent PreparatiON see ss ss esse ee ee 11 Reagent Preparation NoteS ies ss sees ee 12 Assay Procedure aW fees ss se EE ER EER EER EG Ed Re 13 Assay Procedure NoteS ss ee ER RR RE RE ee ee eed 14 Assay Procedure SumMAaFY esse sees sesse ese 16 Calculation of ResultS sesse sesse ee ee Ee AA 17 Troubleshooting Guide ee RR RR RR ER RA ee ee 18 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES ASSAY SPECIFICATIONS Target CTSS Cathepsin S Synonyms CTSS Cathepsin S CTSS cathepsin S
8. ant 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 tissues to a powder with liquid nitrogen Resuspend the powder in 3x 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 then 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
9. centrifuged for 6 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 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 Finely mince tissues and homogenize them in 5 10mL of PBS with a glass homogenizer on ice 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 particu
10. come 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 On disposal flush with large volumes of water to prevent accumulation Returns Refunds Cancelations Any problems with our products must be reported to us within 10 days of product receipt The customer must obtain written authorization from us before returning items To request that goods be returned please contact our Technical Support If an error by MyBioSource Inc results in shipment of an incorrect order we 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 our 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
11. enzyme resulting in color development A sulfuric acid stop solution is added 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 2 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES H gt 3 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES ASSAY PRINCIPLE IMAGE KIT COMPONENTS Component Quantity Coated 96 well Strip Plate 1 Standard Lyophilized 2 vials Sample Diluent 1 vial x 20 ml Assay Diluent A 1 vial x 12 ml Assay Diluent B 1 vial x 12 ml Detection Reagent A 1 vial x 120 ul Detection Reagent B 1 vial x 120 ul Wash Buffer 30x 1 vial x 20 ml TMB Substrate 1 vial x 9 ml Stop Solution 1 vial x 6 ml Adhesive Plate Sealers 4 Instruction Manual 1 KIT STORAGE Upon receipt the kit should be stored at 4 C if intended for use within 24 hours Otherwise the Assay Plate Standard Detection Reagent A and Detection Reagent B should be stored at 20 C Avoid repeated freeze thaw cycles Store all other kit components at 4 C The Substrate should never be frozen Once individual reagents are opened it is recommended that the kit be used within 1 month Unused Strip Plate wells should be stored at
12. ing 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 particular experiments We are 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 8 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES SAM PLE PREPARATION The resulting Optical Density OD values of your sample must fall within the OD values of the standard curve in order for the calculated antigen concentration to be accurate In many cases samples will need to be diluted in order to lower the antigen concentration to sufficient levels Information about antigen concentrations within various sample types may be available from the published literature however it is often necessary to run a dilution series of each sample type The following will prepare sufficient volumes to run the Sample dilution series in triplicate In the case of small volume samples the first step in the series can be a dilution like 1 5 or 1 10 rather than undiluted sample Running duplicate or triplicate wells for each sample is recommended Always dilute samples in the same buffer as the Standard used to generate the Standard Curve Transfer Volume 25ul 25ul 25ul 25ul 25ul 25ul 25ul 250ul Sample Diluent 0 ul 225 u
13. l 225 ul 225 ul 225 ul 225 ul 225 ul 225 ul Dilution 1 1 1 10 1 100 1 1K 1 10K 1 100K 1 1 000K 1 10 000K Special Cases Serum plasma samples require about a 2 fold dilution 9 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES STANDARD 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 10 000 pg ml Reconstitute 1 tube of lyophilized Standard with 1 0 ml of Sample Diluent Incubate at room temperature for 10 minutes with gentle agitation avoid foaming D1 5000 pg ml Pipette 250ul of Stock Standard into 250ul of Sample Diluent D2 2500 pg ml Pipette 250ul of D1 into 250ul of Sample Diluent D3 1250 pg ml Pipette 250ul of D2 into 250ul of Sample Diluent D4 625 pg ml Pipette 250ul of D3 into 250ul of Sample Diluent D5 312 5 pg ml Pipette 250ul of D4 into 250ul of Sample Diluent D6 156 3 pg ml Pipette 250ul of D5 into 250ul of Sample Diluent D7 78 15 pg ml Pipette 250ul of D6 into 250ul of Sample Diluent Zero Standard 0 pg ml Use Sample Diluent alone D7 Zero Standard D1 D2 S
14. late 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 7 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES SAM PLE COLLECTION NOTES 1 We 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 in the results Due to chemical interference the use of tissue or cell extraction samples prepared by chemical lysis buffers may result in inaccurate results Due to 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 heat
15. lor developing time Please perform the experiment exactly according to the supplied instructions 13 Due to inter and intra assay variability it is recommended that appropriate carry over controls be included between assays 14 Prior to running valuable samples we recommend that the user run a preliminary experiment using the supplied controls in order to validate the assay 15 To minimize external influence on the assay performance operational procedures and lab conditions such as room temperature humidity 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 16 The kit should not be used beyond the expiration date on the kit label 15 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES ASSAY PROCEDURE SUMMARY Prepare all reagents samples and standards Add 100 ul standard or sample to each well and incubate for 2 hours at 37 C Aspirate and add 100 ul prepared Detection Reagent A and incubate of 1 hour at 37 C Aspirate and wash 3 times Add 100 ul prepared Detection Reagent B and incubate for 1 hour at 37 C at 37 C 16 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 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 b
16. ng 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 wall or 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 less than 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 12 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 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 100ul of Standard Blank or Sample per well cover with a plate sealer and incubate for 2 hours at 37 C 2 Aspirate the liquid of each well
17. rect assay temperature Briefly spin the vial of standard and dissolve the powder thoroughly by a gentle mix Completely aspirate wells between steps Ensure sufficient incubation time Use recommended incubation temperature Bring substrate to room temperature before use Inadequate reagent volumes Improper dilution Check pipettes and 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 18 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Troubleshooting Guide continued Problem Possible Cause Solution 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 Make 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 19 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Important Note During shipment small volumes of product will occasionally be
18. res 6 Washing Proper washing procedure is critical Insufficient washing will result in poor precision and falsely elevated absorbance readings Residual liquid in the reaction wells should be patted dry against absorbent paper during the washing process Do not put absorbent paper directly into the reaction wells 7 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 8 Reading The microplate reader should be preheated and programmed prior to use Prior to taking OD readings remove any 14 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES residual liquid or fingerprints from the underside of the plate and confirm that there are no bubbles in the wells 9 Reaction Time Control Control reaction time should be strictly followed as outlined 10 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 11 Mixing 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 12 Kits from different batches may be a little different in detection range sensitivity and co
19. tock 10 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES REAGENT PREPARATION Bring all reagents to room temperature 18 25 C before use Detection Reagent A and B Use the Detection Reagent A and B stocks to prepare sufficient volumes of Detection Reagent A and B Working Solution for the number of wells you are planning to run Dilute Detection Reagent A and B to a ratio of 1 100 using Assay Diluent A and B respectively Wash Buffer If crystals have formed in the concentrate warm to room temperature and mix it gently until crystals have completely dissolved Prepare 600 ml of Working Wash Buffer by diluting the supplied 20 ml of 30x Wash Buffer Concentrate with 580 ml of deionized or distilled water Wash Buffer can be stored at 4 C once prepared TB 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 11 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES REAGENT PREPARATION NOTES 1 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 at 20 80 C In order to avoid repeat freeze thaws prepare one time use aliquots of standard for lo
20. y reducing the data using computer software capable of generating a four parameter logistic 4 PL curve 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 these calculations 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 6000 5000 4000 3000 we 2000 rs f ET ai o A z EE a NE o 0 5 1 1 5 2 2 5 Concentration pg mi Optical Density 17 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES TROUBLESHOOTING GUIDE Problem Possible Cause Solution Poor standard curve Low signal Inaccurate pipetting Check pipettes Improper standard dilution Wells not completely aspirated Too brief incubation times Incor
Download Pdf Manuals
Related Search