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1. C CY 8064 15 Version 1207102. evated values for wells containing no sample indicate insufficient 2 Poor duplicates accompani i etailed Protocol were followed accurately such results indicate a washing If all instruct need for washer m 3 Overall low si ainten Oy that desiccation of the plate has occurred between the final wash and addition of Su t gent Do not allow the plate to dry out Add Substrate Reagent immediately after wash amp Y C CY 8064 10 Version 120710 S100A14 ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Reagent Stability All of the reagents included in the CycLex Research Product CircuLex S100A14 ELISA Kit been tested for stability Reagents should not be used beyond the stated expiration date Upon recei reagents should be stored at 4 C except the reconstituted human S100A14 Standard must below 70 C Coated assay plates should be stored in the original foil bag sealed by the containing a desiccant pack Assay Characteristics 1 Typical standard curve be S TORATI prengan ra a A450 0 5 10 15 20 100A14 conc ng ml C CY 8064 11 Version 120710 S100A14 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 2 Precision Intra assay Precision Precision within an assay Three samples of known concentration were tested sixteen times on one plate t
3. For Research Use Only Not for use in diagnostic procedures References Pietas A Schluns K Marenholz I Schafer B W Heizmann C W Petersen I Mole cloning and characterization of the human S100A14 gene encoding a novel member of the S10 family Genomics 79 513 522 2002 2 Adam P J Boyd R Tyson K L Fletcher G C Stamps A Hudson L Poyser N Griffiths M Steers G Harris A L Patel S Berry J Loader J A Townsend L Legrain P Parekh R Terrett J A Comprehensive proteomic analysis of breast membranes reveals unique proteins with potential roles in clinical cancer J Biol Chem 278 6482 6489 2003 3 Wang HY Zhang JY Cui JT Tan XH Li WM Gu J Lu YY Expressio tus S100A14 and S 100A4 correlates with metastatic potential and clinical outcome in colorec after surgery Oncol Rep 23 45 52 2010 Related Products CircuLex S100A13 ELISA Kit Cat CY 8057 CircuLex S100A12 ELISA Kit Cat CY 8058 CircuLex S100P ELISA Kit Cat CY 8060 CircuLex S100A8 MRP8 ELISA Kit Cat CY 8061 CircuLex S100A9 MRP14 ELISA Kit Cat CY 8062 CircuLex S100A11 ELISA Kit Cat CY 8063 CircuLex S100A14 ELISA Kit Cat CY 8064 CircuLex S100A7 Psoriasin ELISA Kit Cat 3 CircuLex S100A4 ELISA Kit Ver 2 Cat CY 8 Anti Human S100A3 Clone YK 3E3 C M1039 Anti Human S100A4 p9Ka Cat CY P Anti Human S100P Cat CY P1028 Anti Human S100A10 Cat
4. Biological samples may be co wounds or breathe aerosols d with infectious agents Do not ingest expose to open tective gloves and dispose of biological samples properly e CAUTION Sulfuric A rong acid Wear disposable gloves and eye protection when handling Solution Q C CY 8064 6 Version 120710 a S100A14 ELISA Kit Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation Collection and Storage Cell culture supernatant conditioned media Remove any particulates by centrifugation and assay immediately or aliquot and store sam below 70 C Avoid repeated freeze thaw cycles Cell lysates 1 Harvest and pellet cells by centrifugation using standard methods 2 Resuspend the cell pellet with an appropriate extraction buffer for example 2 EPES KOH pH 7 5 250 mM NaCl 0 1 NP 40 2 mM CaCl 1 mM EDTA 0 2 mM PMS L pepstatin 0 5 ug mL leupeptin 0 5 mM DTT and lyse the resuspended cells Homogenizer sonication or three cycles of freezing and thawing er a Dounce 3 Transfer extracts to microcentrifuge tubes and centrifuge at 15 000 rpm inutes at 4 C 4 Aliquot cleared lysate to a clean microfuge tube These samples ar to the instructions in Assay Procedure at p 9 y for analysis according NOTE THE ABOVE PROCEDURES ARE INTENDE S A GUIDELINE THE OPTIMAL EXPERIMENTAL CONDITIONS DEPENDING ON THE PARAMETERS BEING INVESTIGATED AND BE
5. 450 nm if only a single can be used Wells must be read within 30 minutes of adding the Stop Solution Note 1 Complete remo remove any te id at each step is essential to good performance After the last wash ing Wash Buffer by aspirating or decanting Invert the plate and blot it againstclea Pg er towels Note 2 Relia fl rves are obtained when either O D values do not exceed 0 2 units for the Sntration or 2 5 units for the highest standard concentration Note 3 If the mi te reader is not capable of reading absorbance greater than the absorbance of the i ndard perform a second reading at 405 nm A new standard curve constructed alues measured at 405 nm is used to determine human S100A14 concentration of CY 8064 Version 120710 S100A14 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Calculations Average the duplicate readings for each standard control and sample and subtract the average standard optical density Plot the optical density for the standards versus the concentration th standards and draw the best curve The data can be linearized by using log log paper andere analysis may be applied to the log transformation To determine the human S100A14 co each sample first find the absorbance value on the y axis and extend a horizontal line t curve At the point of intersection extend a vertical line to the x axis and read the correspo S100A 1
6. DETERMINED BY THE INDIVIDUAL USER NO WARRANT NTEE OF PERFORMANCE USING THESE PROCEDURES IS M Other biological samples Remove any particulates by centrifugation and assayMimmediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycle C CY 8064 7 Version 120710 S100A14 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex S100A14 ELISA Kit is provided with removable stri wells so the assay can be carried out on separate occasions using only the number of strips requi the particular determination Since experimental conditions may vary an aliquot of the hu Standard within the kit should be included in each assay as a calibrator Disposable pi reagent troughs should be used for all liquid transfers to avoid cross contamination samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r are supplied ready to use with the exception of 10X Wash Buffer and Human S100A14 Stand 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X fer to 900 mL of deionized distilled water ddH O Mix well Store at 4 C for two o C for long term storage Prepare Standard Solutions as follows 2 Reconstitute Human S100A14 Standard with 0 85 mL of ddH centration of the human S100A14 in vial should be 64 ng mL which is refe
7. Version 120710 S100A14 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction 100 proteins form a growing subfamily of proteins related by Ca binding motifs to the EF Ca binding protein superfamily By analyzing a human lung cancer cell line subtractive cDNA li Pietas et al identified and characterized S100A14 which encodes a deduced 104 amino aesds ovary breast and uterus tumors and under expression in kidney rectum and cold suggesting distinct regulation with potentially important functions in malignant sformation Using a proteomics approach to identify genes upregulated in breast can wed by database analysis and PCR of a breast carcinoma cell line Adam et al cloned hich they called BCMP84 BCMP84 expression in normal tissue was restricted to stratified s aous epithelium of skin cervix and tonsil Weak staining was seen in normal breast ductal tissue rong immunoreactivity was detected in 10 of 58 17 breast cancer tissues BCMP84 was e n the plasma membrane of transiently transfected breast carcinoma cells 2 C CY 8064 2 Version 120710 S100A14 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CircuLex S100A14 ELISA Kit employs the quantitative sandwich enzyme immuno technique An antibody specific for human S100A14 has been pre coated on
8. 4 concentration If the samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor 1 The dose response curve of this assay fits best to a sigmoidal 5 parameter logistic tion The results of unknown samples can be calculated with any computer program havi eter logistic function It is important to make an appropriate mathematical adjustment mmodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of analyt ration The calibration curve is constructed by plotting the absorbance Y of calib us log of the known concentration X of calibrators using the four parameter fuy Alternatively the logit log function can be used to linearize the calibration curve i e logit ance Y is plotted versus log of the known concentration X of calibrators 3 Dilution factors need to be taken into consideration in calcu e human 100A14 concentration Measurement Range The measurement range is 0 25 ng mL to 16 ng ny sample reading higher than the highest standard should be diluted with Dilution Buff igher dilution and re assayed Dilution factors need to be taken into consideration in calculating t S100A14 concentration Troubleshooting 1 The human S100A14 Standard e run in duplicate using the protocol described in the Detailed Protocol Incubation s mperatures significantly different from those specified may give erroneous results Qi
9. CY P1 Anti Human S100A16 Cat CY P1 Anti Human S100A3 Cat CY Anti Human S100A2 Cat CY4P10 Human S100B Cat CY Human S100A1 Cat CY Human S100A2 Cat Human S100A3 Cat i CY 8064 14 Version 120710 S100A14 ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Human 100A13 Cat CY R2263 Human 8100A14 Cat CY R2264 Human S100A16 Cat CY R2266 Human S100P Cat CY R2267 Human S100A11 Cat CY R2269 Human S100A1 Low Endotoxin Cat CY R2451 O Human S100A3 Low Endotoxin Cat CY R2453 Human S100A4 Low Endotoxin Cat CY R2454 Human S100A7 Low Endotoxin Cat CY R2457 Human S100A8 Low Endotoxin Cat CY R2458 Human S100A9 Low Endotoxin Cat CY R2459 G Human S100A11 Low Endotoxin Cat CY R2461 Human S100A12 Low Endotoxin Cat CY R2462 G Human S100414 Low Endotoxin Cat CY R2464 Human S100P Low Endotoxin Cat CY R2467 QO PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cyclex co jp CycLex CircuLex products are supplied for rese use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval f cLex Co Ltd To inquire about licensing for such commercial use please contact us via G N
10. S100A14 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human S100A14 CircuLex S100A14 ELISA Kit Cat CY 8064 O Intended USE cos ccaresanceaeateiohanecacaessamkedetend 1 LS EE E E A T 1 TACOS OM soisiscainineanncecncaendssnadienneateateneaccccets 2 Principle of the ASSAY cvcsicsssiscsascsccssccavesscdss 3 Materials Provided csiascesscsasssesencesevnenscce 4 Materials Required but not Provided 5 Precautions and Recommendation 6 QO Sample Collection and Storage 0 T Detailed Protocol iisccsccisisvacevesardastennctyvaceree 8 9 CAL CMIAUIONS siaiicecansansietacesaressvenvensxernedenerne 10 Measurement Rative csseccssioissisvceevscesseeeees 10 Troubleshooting scsscsissisciscicessdesivaccesscsegoseree 10 Reagent SCADUNY scaicisccscectsncsuatisnvsivesecesversves 11 Assay Characteristics 11 Example of Test Results eeeeeeneeees Referents i ccissaiseaiisarvicaaveraceiuscdcataieenstonectene 1 Related PrOGUCIS css ssvssncvessssiennenehioavesaacins 14 1 Intended Use The CycLex Research Product Cire A14 ELISA Kit is used for the quantitative measurement of human S100A14 in cell lysate cell culture medium and other biological media This assay kit is for research use olja t for use in diagnostic or therapeutic procedures Storage e Upon receipt store all co e Don t expose reagents t e C CY 8064 1
11. e Sample Preparation above Pipette 100 uL of Standard Solutions Std1 Std7 Blank and appropriately dilut s in duplicates into the appropriate wells 4 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 3 on an orbital microplate shaker 5 Wash 4 times by filling each well with Wash Buffer 350 uL using a s le multi channel pipette manifold dispenser or microplate washer w 6 Add 100 uL of HRP conjugated Detection Antibody into each well N Incubate the plate at room temperature ca 25 C for 1 hour at ca 300 rpm on an orbital microplate shaker 8 Wash 4 times by filling each well with Wash Buffer 350 u pipette manifold dispenser or microplate washer squirt bottle multi channel 9 Add 100 uL of Substrate Reagent Avoid expgSing the microtiter plate to direct sunlight Return Substrate Reagent to 4 C immediately after the sary volume is removed 10 Incubate the plate at room temperature ca 25 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation time maybe extended up to 30 minutes if the reaction temperature is below than 20 C Q 11 Add 100 uL of Stop Solution to e Reagent the same order as the previously added Substrate 12 Measure absorbance in each w a spectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelen of 450 550 or 450 595 nm can also be used Read the microplate at
12. o assess intra precision e Intra assay Within Run n 16 CV 3 7 6 9 Human S100A14 conc ng mL C Sample Sample Sample 3 1 2 3 4 3 6 7 8 3 Linearity To assess the linearity of the assay samp ntaining and or spiked with high concentrations of human S100A14 were serially diluted with i tion Buffer to produce samples with values within the dynamic range of the assay A Linearity 25 pee aaa aera anne Sample 1 Sample 2 ae Sample3 _ oa hu S100A14 cone ng ml o oa o had o 0 0 2 0 4 0 6 0 8 1 1 2 Dilution Ratio C CY 8064 12 Version 120710 S100A14 ELISA Kit TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Human S100A 14 concentration in several cell line conditioned media Concentration of 100A14 in several cell line conditioned media 1 2 1 0 0 8 0 6 0 4 100A14 conc ng ml 0 2 0 0 Lovo A431 KATO Cell line Fig 2 Human 100A 14 concentration in several ce 5x 10 cells ml 100A14 concentration in several cell lysates 3 5 3 0 2 5 2 0 1 5 1 0 100A14 conc ng ml 0 5 0 0 Lovo A431 KATOIN Cell line C CY 8064 13 Version 120710 S100A14 ELISA Kit n TM CircuLex User s Manual
13. quality e Disposable paper towels C CY 8064 5 Version 120710 S100A14 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations e Allow all the components to come to room temperature before use e All microplate strips that are not immediately required should be returned to the zip lock h must be carefully resealed to avoid moisture absorption e Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit e Rinse all detergent residue from glassware Qy e Use deionized water of the highest quality e Do not mix reagents from different kits The buffers and reagents used in this kit contain NaN as preservati hould be taken to avoid direct contact with these reagents Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the N where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containi ions in compliance with local regulations e Avoid contact with Substrate Solution which contains ogen peroxide e Avoid contact with Stop Solution which co Ifuric Acid e Wear gloves and eye protection when and these reagents In case of contact thoroughly with water and seek medi in unodiagnostic materials and samples of rat origin e Stop Solution and the Substrate Solution wash skin on when necessary
14. rred as a Ma ard of human S100A14 Use the Master Standard to produce a dilution series 4 each tube thoroughly before the next transfer The 16 ng mL standard Std 1 serve e highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard ution Buffer Concentration Std 1 150 uL of Master Standard 450 uL 16 ng mL Std 2 300 uL of Std 1 16 ng mL 300 uL 8 ng mL Std 3 300 uL of Std 2 8 ng mL 300 uL 4 ng mL Std 4 300 uL of Std 3 4 ng 300 uL 2 ng mL Std 5 300 uL of Std 4 2 ngAnL 300 uL 1 ng mL Std 6 300 uL of Std 5 1 300 uL 0 5 ng mL Std 7 300 uL of Std 6 0 mL 300 uL 0 25 ng mL Blank 300 uL 0 ng mL Note Do not use a Repe Qy Change tips for every dilution Wet tip with Dilution Buffer before dispensing rtions of Master Standards should be aliquoted and stored at below 70 C immediate void multiple freeze and thaw cycles Sample Diluti e Cell culture tants may not require dilution e Otherbi al Samples require 1 10 fold or appropriate dilution CY 8064 8 Version 120710 S100A14 ELISA Kit n TM CircubLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Procedure paa Remove the appropriate number of microtiter wells from the foil pouch and place them into the holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 Dilute samples with Dilution Buffer Se
15. te One microplate supplied ready to use with 96 wells 12 strips of 8 wells in bag with a desiccant pack Wells are coated with anti S100A14 antibody as a capture antib Human S100A14 Standard One vial containing 54 4 ng of lyophilized recom HRP conjugated Detection Antibody One bottle containing 12 mL of adish peroxidase conjugated anti S 100A 14 antibody Ready to use Substrate Reagent One bottle containing 20 mL of the chromogens e tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL of 1 N H2SO RR C CY 8064 4 Version 120710 S100A14 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for sample preparation e Vortex mixer Qy e Microplate washer optional Manual washing is possible but not preferable e Plate reader capable of measuring absorbance in 96 well plates at dual wave s of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used can also be read at a single wavelength of 450 nm which will give a somewhat higher readi Software package facilitating data generation and analysis opti 9 500 or 1000 mL graduated cylinder e Reagent reservoirs e Deionized water of the highest
16. to a microplate St and samples are pipetted into the wells and the immobilized antibody binds any human S100Ash4 After washing away any unbound substances an HRP conjugated antibody specific for h is added to the wells Following a wash to remove any unbound antibody HRP conjugate conjugate is allowed to react with the substrate H O tetramethylbenzidine The reaction 1 addition of acidic solution and absorbance of the resulting yellow product is measured at 450 absorbance is proportional to the concentration of human 100A14 A standard curvesis constructed by plotting absorbance values versus human S100A14 concentrations of calibrators a entrations of unknown samples are determined using this standard curve The CircuLex 100A14 ELISA Kit is designed to measure the concentration um cell Lysate conditioned medium and other biological media Summary of Procedure QO Add 100 uL of diluted sample to the wells v Incubate for 1 hour p Wash the wells v Add 100 uL of HRP conju A14 antibody Yy Incu hour at room temp Wash the wells Add 100 uL of so pen Add 100 uL of Kp oF at 450 nm 100A 14 from C i C CY 8064 3 Version 120710 S100A14 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided All samples and standards should be assayed in duplicate The following components are supplie are sufficient for the one 96 well microplate kit Micropla
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