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1. Introduction The UCHLI is also called PARKS or neuronal specific protein gene product 9 5 carboxyl terminal ubiquitin hydrolase regulating ubiquitin dependent signaling pathways r suggested as a tumor suppressor UCHLI is expressed predominantly in neurons 1 representi 2 of total soluble brain protein 2 as well as in testis and ovary In vivo UCHLI has be involved in the regulation of the ubiquitin pool apoptosis learning and memory and mice because of spontaneous intragenic deletions yields phenotypes with neurological defe Mutations in UCHL1 have been discovered in a German family with Parkinson disease PD a point mutation near the active site that changes Ile 93 to Met I93M which caused a ial loss of the catalytic activity of this thiol protease This mutation has been linked to an increas f developing an autosomal dominant form of PD 4 Based on the abundance in the CNS UCHLY has been proposed as a candidate biomarker for brain injury and ischemic strokes It was demo t UCHLI was released from injured neurons and flow into the cerebrospinal fluid and eventu circulating blood 5 Principle of the Assay The CycLex Research Product CircuLex Human UCHL1 E SA t employs the quantitative sandwich enzyme immunoassay technique A antibody specific for hu CHL is pre coated onto a microplate Standards and samples are pipetted into the wells the nobilized antibody binds any human UCHLI present After wa2. Troubleshooting 1 The Human UCHLI Standard should begun licate using the protocol described in the Detailed Protocol Incubation times or temperature significantly different from those specified may give erroneous results 2 Poor duplicates accompanied by elev values for wells containing no sample indicate insufficient washing If all instructions i iled Protocol were followed accurately such results indicate a need for washer maintenan 3 Overall low signal may ndic at desiccation of the plate has occurred between the final wash and addition of Substrate Re after wash t Do not allow the plate to dry out Add Substrate Reagent immediately included in the CycLex Research Product CircuLex Human UCHL1 ELISA Kit stability Reagents should not be used beyond the stated expiration date Upon agents should be stored at 4 C except the reconstituted UCHLI Standard must be stored at ted assay plates should be stored in the original foil bag sealed by the zip lock and desiccant pack CY 8092 Versions 120710 Human UCHLI 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 UCHLI giving absorbance ighe than mean absorbance of blank plus three standard deviations of the absorbance of blank 1 3SD blank is better than 253 pg ml of sample
3. Dilution Buffer is pipetted into blank wells Typical Standard Curve Or Human UCHLI Standard Curve A450 0 5 10 15 20 Human UCHLI Conc ng ml C CY 8092 10 Versions 120710 Human UCHLI ELISA Kit x TM Circu Lex 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 seven times on one plate to assess intrazass precision Intra assay Within Run n 7 CV 4 5 6 0 96 Q Human UCHL concentration ug g total protein No Sample 1 Sample2 Sample 3 1 32 7 59 9 10 3 2 31 1 54 8 9 4 3 28 8 54 6 9 0 4 30 1 61 0 10 5 5 32 6 60 0 10 4 6 31 5 58 8 9 3 Q 7 32 5 32 7 min 28 8 54 6 mean 31 3 58 4 SD 1 5 2 6 CV 4 7 Inter assay Precision Precision between assays Three samples of known concentration were teste precision n four separate assays to assess inter assay nter assay Run to Run n 4 CV 2 Human U centration ug g total protein plel Sample2 Sample 3 30 5 62 5 9 0 30 4 56 0 10 2 30 7 57 6 9 4 4 29 3 53 1 8 9 62 5 10 2 Qy min 29 3 53 1 8 9 mean 30 2 57 3 9 4 SD 0 6 4 0 0 6 Qy CV 2 0 6 9 6 0 Sample cell lysate C CY 8092 11 Version 120710 a Human UCHLI ELISA Kit Circulex User s Manual For Research Use Only Not for use in diagnostic procedures AN 3 Linearity Two bi
4. Wash Buffer One bottle containing 100 of 10X buffer containing 206 Tween 20 Dilution Buffer One bottle contai mL of 1X buffer use for reconstitution of Human UCHL1 Standard and sample dilution Rea Human UCHLI Standard containing 200 ng of lyophilized recombinant human UCHLI HRP conjugated Detecti conjugated anti huma ntibody One bottle containing 12 mL of HRP horseradish peroxidase ntibody Ready to use Substrate Reag TMB Ready to tle containing 20 mL of the chromogenic substrate tetra methylbenzidine Stop sain Rp ottle containing 20 mL of 1 N H5SO Ready to use C CY 8092 3 Version 120710 Human UCHLI ELISA Kit x TM Circu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor Orbital microplate shaker Microcentrifuge and tubes for sample preparation 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 Software package facilitating data generation and analysis opti 9 500 or 1000 mL graduated cylinder Reag
5. with 1 0 mL of Dilution B human UCHLI in vial should be 200 ng mL which is referred UCHLI e concentration of the ter Standard of human Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series OW Mix each tube thoroughly before the next transfer The 20 ng mL standard Std 1 ve the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 100 uL of Master Standard 200 ng 900 uL 20 ng mL Std 2 300 uL of Std 1 20 ng mL 300 uL 10 ng mL Std 3 300 uL of Std 2 10 ng mL Q 300 uL 5 ng mL Std 4 300 uL of Std 3 5 ng m 300 uL 2 5 ng mL Std 5 300 uL of Std 4 2 5 ng mL 300 uL 1 25 ng mL Std 6 300 uL of Std 5 1 2 300 uL 0 63 ng mL Std 7 300 uL of Std 6 L 300 uL 0 31 ng mL Blank 300 uL 0 ng mL Note Do not use a Re before dispensin 70 C immedia p ipette Change tips for every dilution Wet tip with Dilution Buffer nused portions of Master Standard should be aliquoted and stored at below id multiple freeze and thaw cycles Sample Prep Cell lysate o biological samples require 10 to 1 000 fold dilution atant samples require 1 to 100 fold dilution CY 8092 Versions 120710 Human UCHLI ELISA Kit x TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay Procedure for Human UCHL1 Remo
6. Human UCHLI ELISA Kit x TM Circu Lex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human UCHL1 CircuLex Human UCHL1 ELISA Kit e Cat CY 8092 O Tine SING a coccatessucncsnmoieetocantatsecaacoannslesten 1 ENO trat A EPEE Cr NTC CT 1 DIAS io NN PPP DUMP 2 Principle of the Assay 2 3 Materials Provided terrens 3 Materials Required but not Provided 4 Precautions and Recommendations 3 Sample Collection and Storage 6 Detailed PEOIDOOL ent te theta ta eere a ttn Rua 7 8 Coe PAINS s sececiioo tt metis hen gat M MU 9 Measurement Range 9 Troubles DG QU Gy scscasesonennscnssceennstatsasaucnceby 9 Reagent SUaDIliby usse esie ertt ene oben pei 9 Assay Characteristics esesssss 10 Example of Test Results i nu TD 1 Related PrOGUOls s oe eoe rtr Rated aH 14 Intended Use The CycLex Research Product CircuLex UCHL1 ELISA Kit is used for the quantitative measurement of human UCHLI in cell lygate cell culture supernatant and other biological media This assay kit is for research use onia for use in diagnostic or therapeutic procedures Storage Upon receipt store all co Don t expose reagents to C CY 8092 1 Version 120710 Human UCHLI ELISA Kit x TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures
7. an be linearized by using log log paper and n analysis may be applied to the log transformation To determine the human UCHLI co n each sample first find the absorbance value on the y axis and extend a horizontal line t dard curve At the point of intersection extend a vertical line to the x axis and read the correspo man UCHLI 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 4 parameter lo c equation The results of unknown samples can be calculated with any computer progr av 4 parameter logistic function It is important to make an appropriate mathematical adjus accommodate for the dilution factor ration The calibration sus log of the known ively the logit log function is plotted versus log of the 2 Most microtiter plate readers perform automatic calculations of analyte curve is constructed by plotting the absorbance Y of calib concentration X of calibrators using the 4 parameter function can be used to linearize the calibration curve i e logit of abs known concentration X of calibrators Measurement Range The measurement range is 0 31 ng mL to 20 standard should be diluted with Dilution Buffer in to be taken into consideration in calculating the hum mple reading higher than the highest dilution and re assayed Dilution factors need HL concentration
8. ent reservoirs Deionized water of the highest quality Disposable paper towels C CY 8092 4 Version 120710 Human UCHLI ELISA Kit x TM Circu Lex User s Manual A For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations Allow all the components to come to room temperature before use All microplate strips that are not immediately required should be returned to the zip lock h must be carefully resealed to avoid moisture absorption Do not use kit components beyond the indicated kit expiration date Use only the microtiter wells provided with the kit Rinse all detergent residues from glassware Qy Use deionized water of the highest quality 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 Do not smoke eat or drink when performing the N where samples or reagents are handled Dispose of tetra methylbenzidine TMB containi ions in compliance with local regulations e Avoid contact with the acidic Stop Solution and Subs Solution which contains hydrogen peroxide e Wear gloves and eye protection when han unodiagnostic materials and samples of human origin and these reagents In case of conta th the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek m at
9. o activation Immediately centrifuge samples at 4 C for 15 minutes at 1 0 store samples on ice for up to 6 hours before assaying Aliquots of 70 C for extended periods of time Avoid repeated freeze thaw cy Note Citrate plasma has not been validated for use in this as Other biological samples Remove any particulates by centri ion and assay immediately or aliquot and store samples at below 70 C Avoid repeated Q yeles C CY 8092 6 Version 120710 Human UCHLI ELISA Kit x TM Circu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CirceuLex Human UCHL1 ELISA Kit is provided with remo strips of wells so the assay can be carried out on separate occasions using only the number o required for the particular determination Since experimental conditions may vary an alique human UCHLI Standard within the kit should be included in each assay as a calibrate pipette tips and reagent troughs should be used for all liquid transfers to avoid cross co reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r ready to use with the exception of 10X Wash Buffer and Human UCHL1 Stan 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X deionized distilled water Mix well Store at 4 C for two weeks or 20 term storage 2 Reconstitute Human UCHL1 Standard
10. ological samples were diluted with Dilution Buffer and assayed after dilution The ne sample is set to 1 4 4 Please note all samples including the neat sample are 10 fold diluted as stat the Assay Procedure The results are summarized in the figure below Linearity 6 c A Sampe1 j 5r Sample 2 gt Human UCHL1 ug g total protein o 1 2 3 4 5 Sample Dilution Ratio 4 Q Sample cell lysate C CY 8092 12 Version 120710 Human UCHLI ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig l Human UCHLI concentrations in several cell culture supernatants A 60 p 50 AE M EE S A E gt 40 F W WM9 A M n 3 I S S Mei DORAE ES leis eoo n 99 z o 2 Ba peeescesesesee4a cec aesoeeeheceo ams e 5 Ei z 10 9 M NM M NM n AL LL B Emmi B IRANRAN N uni D E wn N en e lt aQ a N r 22h BR GSES SEEPS ES EEE EE v x a A s z 5 2 5 E M C CY 8092 13 Versions 120710 Human UCHLI ELISA Kit x TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures References 1 Osaka H Wang YL et al 2003 Ubiquitin carboxy terminal hydrolase L1 binds to and stabi monoubiquitin in neuron Hum Mol Genet 12 1945 58 2 Wilkinson K D Lee K M De
11. shing away any unbound es an HRP conjugated antibody specific for human UCHLI is added to the wells Followi sh to remove any unbound antibody HRP conjugate the remaining conjugate is react with the substrate H O tetramethylbenzidine The reaction is stopped by cidic solution and absorbance of the resulting yellow product is measured at 450 nm orbance is proportional to the concentration of human UCHLI A standard curve is constructed by plotting absorbance values versus human UCHLI concentrations of calibrators and concentrations of u n samples are determined using this standard curve Qy C CY 8092 2 Version 120710 Human UCHLI ELISA Kit x TM Circu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted samples to the wells Y Incubate for 1 hour at room temp e Wash the wells O Add 100 uL of HRP conjugated anti human UCHL1 We Y Incubate for 1 hour at room temp Wash the wells Y Add 100 uL of Substrate Reagent Y Add 100 uL of Stop Solution M Measure absorbance at 450 nm Materials Provided All samples and standards should be assayed in du are sufficient for the one 96 well microplate kit te The following components are supplied and Microplate One microplate supplied ready t ith 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated w uman UCHLI antibody as a capture antibody 10X
12. shpande S Duerksen Hughes P Boss J M Pohl neuron specific protein PGP 9 5 is a ubiquitin carboxyl terminal hydrolase Science 246 6 3 Saigoh K Wang YL et al 1999 Intragenic deletion in the gene encoding ubiquitin carboxy terminal hydrolase in gad mice Nat Genet 23 47 51 4 Leroy E Boyer R et al 1998 The ubiquitin pathway in Parkinson s disease N Q 451 452 5 Papa L Akinyi L et al 2010 Ubiquitin C terminal hydrolase is a novel bi er in humans for severe traumatic brain injury Crit Care Med 38 138 44 Related Products CircuLex Human DJ 1 PARK7 ELISA Kit Cat CY 9050 CircuLex Human 14 3 3 Gamma ELISA Kit Cat CY 8 CycLex Poly Ubiquitinated Protein ELISA Kit Cat C CycLex Poly Ubiquitinated Protein Enrichment amp D CycLex Proteasone Enrichment amp Activity Q PRODUCED BY CycLex Co Ltd Q 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co j URL http www cyclex CycLex CircuL are supplied for research use only CycLex CircuLex products and components the ot be resold modified for resale or used to manufacture commercial products withou r written approval from CycLex Co Ltd To inquire about licensing for such commer e please contact us via email C CY 8092 14 Version 120710
13. sure absorbance in each well usi pectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelen 0 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wa an be used Wells must be read within 30 minutes of adding the Stop Solution liquid at each step is essential to good performance After the last wash g Wash Buffer by aspirating or decanting Invert the plate and blot it towels Note 2 Reliabl ard curves are obtained when either O D values do not exceed 0 25 units for the oncentration or 3 0 units for the highest standard concentration The plate nitored at 5 minute intervals for approximately 30 minutes Note icroplate reader is not capable of reading absorbance greater than the absorbance of the andard perform a second reading at 405 nm A new standard curve constructed he values measured at 405 nm is used to determine human UCHL1 concentration of ff scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Note 1 Complete remoya CY 8092 Versions 120710 Human UCHLI ELISA Kit x TM Circu Lex 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 c
14. tention when necessary Biological samples may be cont ith infectious agents Do not ingest expose to open wounds or breathe aerosols W ective gloves and dispose of biological samples properly handling Sto Q CAUTION Sulfuric i127 g acid Wear disposable gloves and eye protection when n C CY 8092 5 Versions 120710 Human UCHLI ELISA Kit x TM Circu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Cell lysates Prepare cell lysates Assay immediately or store the samples on ice for a few hours b assaying Aliquots of the samples may also be stored at below 70 C for extended periods o e Avoid repeated freeze thaw cycles Cell culture supernatant Remove any particulates by centrifugation and assay immedia quot and store samples at below 70 C Avoid repeated freeze thaw cycles entrifuge the e samples on for extended Serum Use a serum separator tube and allow samples to clot for 60 30 minut samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately ice for up to 6 hours before assaying Aliquots of serum may also be stored at be periods of time Avoid repeated freeze thaw cycles the plasma into a in vitro complement Assay immediately or ay also be stored at below Plasma Collect plasma using EDTA Na as the anticoagulant If possib mixture of EDTA Na and Futhan5 to stabilize the sample against sp
15. ve 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 See Sample Preparation above w Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted samples in dup 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 6 Add 100 uL of HRP conjugated Detection Antibody into each well Incubate the plate at room temperature ca 25 C for 1 hour at ca 300 rpm on an orbital microplate shaker oo Wash 4 times by filling each well with Wash Buffer 350 u pipette manifold dispenser or microplate washer sin squirt bottle multi channel 9 Add 100 uL of Substrate Reagent Avoid expos the plate with e g aluminum foil is recommende eturn Substrate Reagent to 4 C immediately after the necessary volume is removed 10 Incubate the plate at room temperature ca orbital microplate shaker The incubation temperature is below than 20 C ay be extended up to 30 minutes if the reaction 11 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 12 Mea
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