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1. Joosse M van Dongen J W Vanacore N van Swi Brice A Meco G van Duijng C M Oostra B A Heutink P Science 299 256 259 2 n Cookson M R Neuron 37 7 10 2003 6 Mitsumoto A amp Nakagawa Y Free Radical Res 35 885 893 2001 N Canet Aviles R M Wilson M A Miller D W Ahmad R Baptista M J Ringe D Petsko G A amp Cookson M R 9103 9108 2004 C Bandyopadhyay S atl Acad Sci USA 101 oo Taha T Saito Y Niki T Iguchi Ariga S M Takahashi 2004 iga H EMBO Rep 5 213 218 9 Martinat C Shendelrnan S Jonason A Leet ea F Yang L Floss T amp Abeliovich A PLoS Biol 2 e327 2004 10 Yokota T Sugawara K Ito K Takahashi R Ariga H amp Mizusawa H Biochem Biophys Res Commun 312 1342 1348 2003 11 Niki T Takahashi Niki K Taira Spain S M amp Ariga H Mol Cancer Res 1 247 261 2003 12 Takahashi K Taira T Niki T Se lguchi Ariga S M amp Ariga H J Biol Chern 276 37556 37563 2001 13 Hod Y Pentyala S N W amp El Maghrabi M R J Cell Biochem 72 435 444 1999 14 Shendelman S Jcmas imag C Leete T amp Abeliovich A PLoS Biol 2 e362 2004 15 F Le Naour D E Cancer Res 3 C Krause L Deneux T J Giordano S Scholl and S M Hanash Clin 01 16 J P MacKei 6928 6934 C lements J D Lich R M Pope Y Hod and J2. Mesylate to stabilize the sample against spontaneous in vitro complement activation Immediately centrifuge samples at 4 C for 15 minutes 00 x g Assay immediately or store samples on ice for up to 6 hours before assaying Aliquots of may also be stored at below 70 C for extended periods of time Avoid repeated freeze thaw cycl Note Citrate plasma has not been validated for use in this assay for a few hours before ended periods of time Avoid repeated freeze thaw cycles See below Other biological samples Remove any particulates by centrifugati ssay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw s Cell lysate Prepare cell lysates Assay immediately or store the samples 6 assaying Aliquots of the samples may also be stored at below 70 C C CY 9050 7 Version 130326 Human DJ 1 PARK7 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 Human DJ 1 PARK7 ELISA Kit is provided removable strips of wells so the assay can be carried out on separate occasions using only the nu strips required for the particular determination Since experimental conditions may vary angali the human DJ 1 PARK7 Standard within the kit should be included in each assay ag 4 Disposable pipette tips and reagent troughs should be used for all liquid trans cross contamination of reagents or sample
3. P Ting Cancer Res 63 CY 9050 15 Version 130326 Human DJ 1 PARK7 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Related Products Parkinson s disease CircuLex Human UCHL1 ELISA Kit Cat CY 8092 CircuLex Mouse UCHL1 ELISA Kit Cat CY 8093 Anti Human UCHL1 PARKS Clone YK 3G8 Cat CY M1040 o Neurological disorder CircuLex 14 3 3 Gamma ELISA Kit Cat CY 8082 Oxidation PA0122 Pseudomonas aeruginosa Low Endotoxin Cat CY R2474 Asp hemolysin Low Endotoxin Cat CY R2475 z PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 A Japan Fax 81 265 76 761 prior written approval from CycLex Co Ltd To inquire about licensing for use please contact us via email CY 9050 Version 130326
4. td 5 6 25 ng ml 300 uL 3 13 ng mL Std 7 3 td 6 3 13 ng ml 300 uL 1 56 ng mL Blank z 300 uL 0 ng mL Note Do n epeating pipette Change tips for every dilution Wet tip with Dilution Buffer oane Unused portions of Standards should be aliquoted and stored at below 70 C e Avoid multiple freeze and thaw cycles CY 9050 Version 130326 Human DJ 1 PARK7 ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation e Serum and plasma samples may require a 10 fold or more dilution e g 25 uL sample 225 uL Dilution Buffer e Cell lysates and other biological samples might be required 10 to 100 fold dilution O 4 Note Higher dilution of samples is recommended Standard Assay Procedure Remove the appropriate number of microtiter wells from the foil pouch and plac nto the well holder Return any unused wells to the foil pouch refold seal with tape and 2 Dilute samples with Dilution Buffer See Sample Preparation above 3 Pipette 100 uL of Standard Solutions Std1 Std7 Blank and dilut les in duplicates into the appropriate wells 4 Incubate the plate at room temperature ca 25 C for 1 hous at ca 300 rpm on an orbital microplate shaker 5 Wash 4 times by filling each well with Wash Buffer 35 sing a squirt bottle multi channel pipette manifold dispenser or microplate washer 6 Add 100 uL of HRP conjugated Detection
5. 2 1 759 5 939 923 1 606 6 952 1 62 1 091 Inter assay Precision Precision between assays Three samples of known concentration VA in three separate assays to assess inter assay precision e Inter assay Run to Run n 3 CV Samples cell lysates le 1 Sample2 Sample 3 MEAN 1 004 1 046 1 791 Qy S D 72 9 92 7 52 9 C V 7 3 8 9 3 3 C CY 9050 13 Version 130326 Human DJ 1 PARK7 ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 3 Linearity To assess the linearity of the assay three samples were serially diluted with the Dilution Buffer produce samples with values within the dynamic range of the assay Samples cell lysat Linearity Human DJ 1 Park7 conc ng mL 0 4 0 6 Sample Dilution Ratio C CY 9050 14 Version 130326 Human DJ 1 PARK7 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures References 1 D Nagakubo T Taira H Kitaura M Ikeda K Tamai S M Iguchi Ariga and H Ariga Bioc Biophys Res Commun 231 509 513 1997 2 A Wagenfeld J Gromoll and T G Cooper Biochem Biophys Res Commun 251 545 ies Y Hod S N Pentyala T C Whyard and M R El Maghrabi J Cell Biochem 72 435 4 4 Bonifati V Rizzu P van Baren M J Schaap O Breedveld G J Krieger E Dekker M C J Squitieri F Ibanez P
6. An to each well 7 Incubate the plate at room temperature ca 25 C 1 hour shaking at ca 300 rpm on an orbital microplate shaker 8 Wash 4 times by filling each well with ffer 350 uL using a squirt bottle multi channel pipette manifold dispenser or micropl as 9 Add 100 uL of Substrate Reage xposing the microtiter plate to direct sunlight Covering the plate with e g aluminum foilais r ended Return Substrate Reagent to 4 C immediately after the necessary volume is remove 10 Incubate the plate at room ture ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shak ubation time may be extended up to 30 minutes if the reaction temperature is below t 0 C Q ion to each well in the same order as the previously added Substrate each well using a spectrophotometric microplate reader at dual wavelengths of wavelengths of 450 550 or 450 595 nm can also be used Read the microplate at ngle wavelength can be used Wells must be read within 30 minutes of adding the 11 Add 100 uL Reagent 12 Measure ab 450 540 n CY 9050 9 Version 130326 Human DJ 1 PARK7 ELISA Kit n TM CircubLex User s Manual For Research Use Only Not for use in diagnostic procedures remove any remaining Wash Buffer by aspirating or decanting Invert the plate and bl against clean paper towels Note 2 Reliable standard curves are obtained when either O D values do not exceed 0 25 units fo blank zero concentrati
7. Human DJ 1 PARK7 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human DJ 1 PARK7 CircuLex Human DJ 1 PARK7 ELISA Ki Cat CY 9050 O Intended Use isssicsalstatatcssassaaaiaaatalenanonataons 1 AOI aT EEEE 1 TPO CUO a esscasasnceadatacatantanatabotectaceceverdoond 2 3 Principle Of the Assay 3 4 Materials Provided pisscccsessssanccncccsaoniecanceruaneas 4 Materials Required but not Provided 5 Precautions and Recommendations 6 QO Sample Collection and Storage 0 7 Detailed Protocol sascsccscsasssvaransassrevinasyscancars 8 10 CACM LALIOIIS cecsncasaacestesassacnavnessgunenwndserteusde 10 Measurement Rative ccssecsccccarsnsesscacenss 10 Troubleshooting vis ccscccassercecaccsesnsasasanassevenee 11 Reagent SCADIIY vrscccseasas scssuvnrvecancaronsasaacdvays 11 Assay CharacterisStics ccc cssscecacessavsseencessancs 12 REI E CES S Related PROGUCS vasisnivsnceesadsavescasasassunneryeseane 1 quantitative measurement of human DJ 1 P serum plasma cell lysate and other biological Intended Use The CycLex Research Product crea Pg DJ 1 PARK7 ELISA Kit is used for the samples This assay kit is for research use onlypz for use in diagnostic or therapeutic procedures Storage e Upon receipt store all co e Don t expose reagents to C CY 9050 1 Version 130326 Human DJ 1 PARK7 ELISA Kit n TM CircuLex Us
8. d 100 uL of diluted sample to the wells Yy Incubate for 1 hour at room temp cA Wash the wells O v Add 100 uL of HRP conjugated anti DJ 1 antibody v Incubate for 1hour at room temp Wash the wells Add 100 uL of Substrate Reagent y Add 100 uL of Stop Solution v Measure absorbance at 450 nm The CircuLex Human DJ 1 PARK7 ELISA Kit is DJ 1 PARK7 from human serum plasma human ce assay is shown in below Diluted samples and serially are added to an appropriate number of wells of the mic esigned to measure the concentration of human es or conditioned medium The principle of the luted human DJ 1 PARK7 standard solutions Otiter plate and incubated DJ 1 PARK7 in the sample will be bound by the primary anti DJ 7 antibody immobilized in the well first reaction After washing the HRP conjugated anti DJ 7 antibody is added to each well and allowed to incubate second reaction The HRP 6nj anti DJ 1 PARK7 antibody will bind to the DJ 1 PARK7 trapped in the well in the first ction After washing the colorimetric substrate for the enzyme is added to all wells and incuba e color development is terminated by the addition of a stop solution The intensity of the c easured at 450 nm The concentrations of the samples tested are calculated using the absorban lue syof the DJ 1 PARK7 standard solutions assayed at the same time amp Y lt amp Y C CY 9050 3 Version 130326 Human DJ 1 PARK7 ELISA Kit n TM CircuLex U
9. eparation 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 e s of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used The pl so be read at a single wavelength of 450 nm which will give a somewhat higher reading e Software package facilitating data generation and analysis opti pal e 500 or 1000 mL graduated cylinder e Reagent reservoirs Deionized water of the highest quality e Disposable paper towels C CY 9050 5 Version 130326 Human DJ 1 PARK7 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 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 should 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 dri
10. er s Manual For Research Use Only Not for use in diagnostic procedures Introduction DJ 1 PARK7 CAPI RS was originally cloned as a putative oncogene capable of transfo NIH 3T3 cells in cooperation with H ras 1 a protein expressed in sperm 2 and a regul 0 RNA protein interactions 3 DJ 1 has also been isolated as a gene associated with early onset Parkinson s disease PD 4 Taken together DJ 1 appears to be involv d biological processes 5 First several lines of evidence suggest that DJ 1 plays a role i stress response 6 7 In cultured mammalian cells DJ 1 quenches reactive oxygen spe eie converted into a variant with a more acidic isoelectric point 6 8 Therefore DJ 1 prote against reactive oxygen species induced cell death and its suppression with small interfering RNA siRNA sensitizes cells to such insults 7 10 Second DJ 1 modulates transcription thro raction with DJ 1 binding protein 11 as well as with protein inhibitor of activated STAT P The latter i modulates the activity of various transcription factors Third DJ 1 has been r nized s a regulatory subunit of an RNA binding protein 13 Fourth DJ 1 which is structurall to the molecular a chaperone Hsp31 may have chaperone activity itself preventing heat indi ation of substrate proteins including alpha synuclein 14 In addition several lines of evidence suggest that DJ 1 plays a role n tumorigenesis First breast canc
11. er patients have elevated levels of circulating DJ 1 and anti antibodies compared to healthy and non breast cancer patients 15 Secondly DJ 1 protei eased in primary non small cell lung carcinoma samples 16 Thirdly treatment of cells uman lung cancer cell line NCI H157 with paclitaxel and MEK inhibitor U0126 leads to a eas ih DJ 1 protein expression 16 Principle of the Assay The CircuLex Human DJ 1 PARK7 ELISA immunoassay technique An antibody specific for Standards and samples are pipetted into the wells a antibody After washing away any unbound substances added to the wells Following a wash to rem conjugate is allowed to react with the substr addition of acidic solution and absorbanc absorbance is proportional to the conc absorbance values versus DJ 1 concentra determined using this standard curv amp Y N amp o p the quantitative sandwich enzyme DJ 1 has been pre coated onto a microplate ny DJ 1 present is bound by the immobilized HRP conjugated antibody specific for DJ 1 is y unbound antibody HRP conjugate the remaining tetramethylbenzidine The reaction is stopped by ulting yellow product is measured at 450 nm The of DJ 1 A standard curve is constructed by plotting calibrators and concentrations of unknown samples are C CY 9050 2 Version 130326 Human DJ 1 PARK7 ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Ad
12. function can be used to linearize the calib urve i e logit of absorbance Y is plotted versus log of the known concentration X of cali r Measurement Range The measurement range is 1 5 o 100 ng mL Any sample reading higher than the highest standard should be diluted with Di uffer in higher dilution and re assayed Dilution factors need to be taken into consideration i g the human DJ 1 PARK7 concentration Q C CY 9050 10 Version 130326 Human DJ 1 PARK7 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting 1 The Human DJ 1 PARK7 Standard should be run in duplicate using the protocol described i Detailed Protocol Incubation times or temperatures significantly different from those specifi a give erroneous results 2 Poor duplicates accompanied by elevated values for wells containing no sample indicz washing If all instructions in the Detailed Protocol were followed accurately such rest need for washer maintenance addition of Substrate Reagent Do not allow the plate to dry out Add Substrate immediately after wash 3 Overall low signal may indicate that desiccation of the plate has occurred sy wash and e Reagent Stability All of the reagents included in the CycLex Research Product Cir uman DJ 1 PARK7 ELISA Kit have been tested for stability Reagents should not be use nd the stated expiration date Upon receipt kit reage
13. nk when performing the RN where samples or reagents are handled e Dispose of tetra methylbenzidine TMB contai ions in compliance with local regulations e Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide e Wear gloves and eye protection when hai unodiagnostic materials and samples of human origin and these reagents In case of co he Stop Solution and the Substrate Solution wash skin thoroughly with water and seek m l attention when necessary Biological samples may be cont ith infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly handling St Q e CAUTION Sulfuric Sint g acid Wear disposable gloves and eye protection when n C CY 9050 6 Version 130326 Human DJ 1 PARK7 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Serum Use a serum separator tube and allow samples to clot for 60 30 minutes Centrifug samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or store sam o ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 C e periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA Na as the anticoagulant If possible collect the to a mixture of EDTA Na and Futhan5 Nafamostat
14. nts should be stored at 4 C except the stituted Human DJ 1 PARK7 Standard must be stored at below 70 C Coated assay plates s stored in the original foil bag sealed by the zip lock and containing a desiccant pack For research use only not for use in diagnostic or therapeut ocedures C CY 9050 11 Version 130326 Human DJ 1 PARK7 ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of human DJ 1 PARK7 giving absogbanc higher than mean absorbance of blank plus three standard deviations of the absorbance blank 3SD blank is better than 0 55 ng mL of sample Dilution Buffer is pipetted into blank wells Typical standard curve DJ 1 Park7 Standard Curve A450 0 20 40 60 80 100 DJ 1 Park7 conc ng mL C CY 9050 12 Version 130326 Human DJ 1 PARK7 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 six times on one plate to assess intrazass precision e Intra assay Run to Run n 6 CV 3 6 6 2 Sample ates Human DJ 1 PARK7 concentration ng mL Sample 1 Sample 2 Sample 3 1 1 012 1 048 1 838 2 970 1 030 1 573 3 1 031 999 1 631 4 985 1 04
15. ons or 3 0 units for the highest standard concentration The pla should be monitored at 5 minute intervals for approximately 30 minutes Note 3 If the microplate reader is not capable of reading absorbance greater than the absofb Average the duplicate readings for each standard control and sample and s average zero standard optical density Plot the optical density for the standards versus Oncentration of the standards and draw the best curve The data can be linearized by using log logspaper and regression concentration of each to the standard curve corresponding DJ 1 PARK7 the standard curve must be sample first find the absorbance value on the y axis and extend a horizo At the point of intersection extend a vertical line to the x axis and re concentration If the samples have been diluted the concentration multiplied by the dilution factor 1 The dose response curve of this assay fits best to a sigg oi p eter logistic equation The results of unknown samples can be calculated with er program having a 5 parameter logistic function It is important to make an appropsiate hematical adjustment to accommodate for the dilution factor 2 Most microtiter plate readers perform automatic c ations of analyte concentration The calibration curve is constructed by plotting the absorbance of calibrators versus log of the known concentration X of calibrators using th ur parameter function Alternatively the logit log
16. s Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay Gy supplied ready to use with the exception of 10X Wash Buffer 20X HRP conjugated Dete ntibody and Human DJ 1 PARK7 Standard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10 deionized distilled water ddH O Mix well Store at 4 C for two aS fer to 900 mL of or 20 C for long term storage 2 Prepare HRP conjugated Detection Antibody by 20 fold diluting the RP conjugated Detection Antibody with Conjugate Dilution Buffer at the time of assa Prepare appropriate volume for your assay Discard unused HRP conjugated Detection Antibody after diluted 3 Reconstitute Human DJ 1 PARK7 Standard wi human DJ 1 PARK7 in vial should be 3 5 DJ 1 PARK7 ddH 0 The concentration of the erred as a Master Standard of human Prepare Standard Solutions as follows Use the Master Standard to produce a dihution series below Mix each tube thoroughly before the next transfer The 100 ng mL stan d 1 serves as the highest standard The Dilution Buffer serves as the zero standard Bla Volume of St Dilution Buffer Concentration Std 1 20 uL of Master 680 uL 100 ng mL Std 2 300 uL of Std ml 300 uL 50 ng mL Std 3 300 uL of g ml 300 uL 25 ng mL Std 4 300 uL o 5 ng ml 300 pL 12 5 ng mL Std 5 300 u 12 5 ng ml 300 uL 6 25 ng mL Std 6 300 uL
17. ser 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 Microplate 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 human DJ 1 PARK7 antibod antibody 10X Wash Buffer One 100 mL bottle of 10X buffer containing 2 Tween 20 Q Dilution Buffer One bottle containing 50 mL of 1X buffer use for sample dilution o use Human DJ 1 PARK7 Standard One vial containing 3 5 ug of lyophilized hu 1 PARK7 20X HRP conjugated Detection Antibody One vial containing of HRP horseradish peroxidase conjugated anti human DJ 1 PARK7 antibody Conjugate Dilution Buffer One bottle containing 12 mL of Conjug tion Buffer Substrate Reagent 20 mL of the chromogenic substrate tetra enzidine TMB Ready to use Stop Solution One bottle supplied ready to use containing 2 of 1 N H2SO4 C CY 9050 4 Version 130326 Human DJ 1 PARK7 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 pr
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