CY-8093 Mouse UCHL1 ELISA Kit
Contents
1. User s Manual For Research Use Only Not for use in diagnostic procedures Introduction The UCHLI also called neuronal specific protein gene product 9 5 is a carboxyl terminal ubiquitin hydrolase regulating ubiquitin dependent signaling pathways recently suggested as a tumor suppressor UCHLI is expressed predominantly in neurons 1 representing to 2 of total soluble brain protein 2 as well as in testis and ovary In vivo UCHLI has been shown to be involved in the egulation of the ubiquitin pool apoptosis learning and memory and its absence in mice because of spontaneous intragenic deletions yields phenotypes with neurological defects 3 Mutations in UCHLh have been discovered in a German family with Parkinson disease PD a point mutation near the active site that changes Ile 93 to Met 193M which caused a partial loss of the catalytic activity of this thiol protease This mutation has been linked to an increased risk of developing an autosomal domigant form of PD 4 Based on the abundance in the CNS UCHLI has been proposed as a candidate biomarker for brain injury and ischemic strokes It was demonstrated that UCHLI was released from injured neurons and flow into the cerebrospinal fluid and eventually into circulating blood 5 Principle of the Assay The CycLex Research Product CircuLex Mouse UCHL1 ELISA Kit employs the quantitative sandwich enzyme immunoassay technique An antibody specific foxymouse UCHLI is pre coat2. room temperature ca 25 C for 1 hour shaking at ca 300frpim on an orbital microplate shaker n Wash 4 times by filling each well with Wash Buffer 350 uL using a sqtirt bottle multi channel pipette manifold dispenser or microplate washer 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 shaking at ca 300 rpm on an orbital microplate shaker oo Wash 4 times by filling each well with Wash Buffer 350 tL using a squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent Avoid exposifig the microtiter plate to direct sunlight Covering the plate with e g aluminum foil is recommended Return Substrate Reagent to 4 C immediately after the necessary volume is removed 10 Incubate the plate at room temperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation Aim may be extended up to 30 minutes if the reaction temperature is below than 20 C 11 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 12 Measure absorbance in each wellUsing aspectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wayelength can be used Wells must be read within 30 mi
3. Lex Research Product CircuLex Mouse UCHL1 ELISA Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Since experimental conditions may vary an aliquot of the mouse UCHLI Standard within the kit should be included in each assay as a calibratot Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r agents are supplied ready to use with the exception of 10X Wash Buffer and Mouse UCHLI Standard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer provided to 900 mL of deionized distilled water Mix well Store at 4 C for two weeks Of 20 C for long term storage 2 Reconstitute Mouse UCHL1 Standard with 1 0 mL of DilutionsBuffer The concentration of the mouse UCHLI in vial should be 100 ng mL which is referred as a Master Standard of mouse UCHLI Prepare Standard solutions as follows Use the Master Standard to produce a dilution series below Mix each tube thoroughly before the next transfer The 20 ng mL standard Stdeb serves jas the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Conc
4. P i Mouse UCHLI ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Mouse UCHL1 Yy CircuLex Mouse UCHL1 ELISA Kit N Cat CY 8093 oO Intended Use 1 gt trat A eee E ene een arr ern eee 1 Introd uctiOn cceceeeesesseeeeseseeeeseteeeessnees 2 Principle of the Assay 2 3 Materials Provided c cccecscccesesseeeeeeees 3 Materials Required but not Provided 4 Precautions and Recommendation 5 Sample Collection and Storage 6 Detailed Protocol ccceecesceeesssceeeeseeeeeeees 7 8 Calculations cccescsscceeseceeeese ec esseeeeeeaes 9 Measurement Range 9 Troubleshooting isssccssecsseacsssccesacasssssoevaccasas 9 Reagent SAD cccczsisssitatonssvorccatadacsamaaseerse 9 Assay Characteristics cccccsscsssceeseeees 10 Example of Test Results ssissisiiscsssscceteasonseveece 1 ReferenCeSixi iisscesesesccesessedeet scededeesteavessiedesaeys Related Products c cccesesceeessreeeesseteeees 14 Intended Use Cs The CycLex Research Product CircuLex UCHL1 ELISA Kit is used for the quantitative measurement of mouse UCHLI in cell ly c ture supernatant and other biological media This assay kit is for research use o Q for use in diagnostic or therapeutic procedures Storage e Upon receipt store all co e Don t expose reagents to C CY 8093 1 Version 140602 Mouse UCHLI ELISA Kit L ircuLex
5. ank is better than 229 pg ml of sample Dilution Buffer is pipetted into blank wells Typical Standard Curve 2 5 p Mouse UCHL1 Standard Curve A450 0 5 10 15 20 Mouse UCHL1 Conc ng ml v CY 8093 10 Version 140602 Mouse UCHL1 ELISA Kit CircuLex Ue Manual For Research Use Only Not for use in diagnostic procedures 2 Precision Intra assay Precision Precision within an assay A sample of known concentration was tested seven times on one plate to assess intra assay precisi e Intra assay Within Run n 7 CV 5 7 Mouse UCHL1 concentration pg mg total protein No Sample 1 1 796 8 2 705 4 3 724 1 4 790 5 5 801 3 6 713 8 7 724 3 max 801 3 min 705 4 mean 750 9 SD 43 0 CV 5 7 Inter assay Precision Precision between assays A sample of known concentration was tested in three arate assays to assess inter assay precision e Inter assay Run to Run n 3 CV 0 7 Mouse UCHL1 c ntration pg mg total protein Sample 1 764 2 754 2 Q 3 758 0 A max 7642 min 754 2 mean 758 8 Qy SD 5 0 CV 0 7 C CY 8093 11 Version 140602 Mouse UCHL1 ELISA Kit CircuLex Ue Manual For Research Use Only Not for use in diagnostic procedures 3 Linearity One biological sample were diluted with Dilution Buffer and assayed after dilution The neat sam is set to 1 3 3 Please note a neat sample is 3 fold diluted as stated in the Assa
6. ed onto a microplate Standards and samples are pipetted into the wells an thejimmobilized antibody binds any mouse UCHLI present After washing away any unbound substances jan HRP conjugated antibody specific for mouse UCHLI is added to the wells Following a wash to remove any unbound antibody HRP conjugate the remaining conjugate is allowed toy react with the substrate H202 tetramethylbenzidine The reaction is stopped by additionjof acidic solution and absorbance of the resulting yellow product is measured at 450 nm The absorbance is proportional to the concentration of Mouse UCHLI A standard curve is constructed 6y plotting absorbance values versus mouse UCHL1 concentrations of calibrators and concentrations of unknown samples are determined using this standard curve Cat CY 8093 2 Version 140602 Mouse UCHL1 ELISA Kit CircuLex Ue Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted samples to the wells 4 Incubate for 1 hour at room temp Wash the wells O Add 100 uL of HRP conjugated anti mouse UCHLI antibody 4 Incubate for 1 hour at room temp Wash the wells Add 100 uL of Substrate Reagent 4 Add 100 uL of Stop Solution O t Measure absorbance at 450 nm Materials Provided All samples and standards should be assayed in The following components are supplied and are sufficient for the one 96 well microplate kit Microplate One microplate su
7. entration Std 1 120 uL of Master Standard 200 ng mL 480 uL 20 ng mL Std 2 300 pL of Std 1 20 ng mL 300 pL 10 ng mL Std 3 300 uL of Std 2 10 ng mL 300 uL 5 ng mL Std 4 300 uL of Std 3 5 ng mL 300 uL 2 5 ng mL Std 5 300 pL of Std 4 2 5 ng mb 300 uL 1 25 ng mL Std 6 300 pL of Std 5 1 25 ngimL 300 uL 0 63 ng mL Std 7 300 uL of Std 6 0 625 ng mL 300 uL 0 31 ng mL Blank 7 300 uL 0 ng mL Note Do not use a Repeating pipette Change tips for every dilution Wet tip with Dilution Buffer before dispensing Unused portions of Master Standard should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Sample Preparation e Cell lysates and other biological samples might be required 10 to 100 fold dilution e Cell culture supernatants may not require dilution Cat CY 8093 T Version 140602 Mouse UCHLI ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay Procedure for Mouse UCHL1 j Remove the appropriate number of microtiter wells from the foil pouch and place them into the well 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 Mouse UCHL1 Standards Std1 Std7 Blank and diluted samples in duplicates into the appropriate wells ENS Incubate the plate at
8. igher reading Software package facilitating data generation and analysis opti 500 or 1000 mL graduated cylinder e Reagent reservoirs Deionized water of the highest quality e Disposable paper towels C CY 8093 4 Version 140602 Mouse UCHLI ELISA Kit L 1rcu Lex 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 pouch Which 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 residues from glassware e Use deionized water of the highest quality e Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or othetchemiucals Care should be taken to avoid direct contact with these reagents e Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the assay or in argas where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containifg solutions 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 handlingjimmu
9. licate using the protocol described in the Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results 2 Poor duplicates accompanied bypelevated values for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed accurately such results indicate a need for washer maintenang 3 Overall low signal may sndicateithat desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash Reagent Stability All of the r agents included in the CycLex Research Product CircuLex Mouse UCHL1 ELISA Kit have beem tested for stability Reagents should not be used beyond the stated expiration date Upon receipt k t reagents should be stored at 4 C except the reconstituted UCHL1 Standard must be stored at below 70 CmGoated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack Cat CY 8093 9 Version 140602 P i Mouse UCHLI ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures L Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of mouse UCHLI giving absorbance than mean absorbance of blank plus three standard deviations of the absorbance of blank A b 3SD bl
10. nodiagnostic materials and samples of human origin and these reagents In case of contaetywith the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medieal att ntion when necessary Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly e CAUTION Sulfuric Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 8093 5 Version 140602 Mouse UCHL1 ELISA Kit CircuLex Uer Manual 4 For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Cell lysate 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 ofgtim Avoid repeated freeze thaw cycles See below Cell culture supernatant Remove any particulates by centrifugation and assay immedia aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles Other biological samples Remove any particulates by centrifugation and assay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles C CY 8093 6 Version 140602 Mouse UCHLI ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The Cyc
11. nutes of adding the Stop Solution Note 1 Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against Clean pap r towels Note 2 Reliable standard curves are obtained when either O D values do not exceed 0 25 units for the blank zetoyconcentration or 3 0 units for the highest standard concentration The plate shouldbe monitored at 5 minute intervals for approximately 30 minutes Note 3 Jf the micfoplate reader is not capable of reading absorbance greater than the absorbance of the highestystandard perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determine mouse UCHLI concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Cat CY 8093 8 Version 140602 Ti Mouse UCHLI ELISA Kit L 1rcu 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 zero standard optical density Plot the optical density for the standards versus the concentration of the standards and draw the best curve The data can be linearized by using log log paper and_regression analysis may be applied to the log transformation To determine the mouse UCHLI concentration of each sam
12. ple first find the absorbance value on the y axis and extend a horizontal line to the standard curve At the point of intersection extend a vertical line to the x axis and read the corresponding mouse 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 logistic quation The results of unknown samples can be calculated with any computer program having a 4 parameter logistic function It is important to make an appropriate mathematical adjustment t0 accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of analyte concentration The calibration curve is constructed by plotting the absorbance Y of calibrators wersus log of the known concentration X of calibrators using the 4 parameter function Alternatively the logit log function can be used to linearize the calibration curve i e logit of absorbance Y is plotted versus log of the known concentration X of calibrators Measurement Range The measurement range is 0 31 ng mL to 20 ng ml Any Sample reading higher than the highest standard should be diluted with Dilution Buffer in higher dilution and re assayed Dilution factors need to be taken into consideration in calculating the mouse UCHLI concentration Troubleshooting 1 The Mouse UCHLI Standard should be runinmdup
13. pplied ready to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated wi i mouse UCHLI antibody as a capture antibody 10X Wash Buffer One bottle containin 10X buffer containing 2 Tween 20 Dilution Buffer One bottle containi of 1X buffer use for reconstitution of Mouse UCHL1 Standard and sample dilution Read Mouse UCHL1 Standard On aining 100 ng of lyophilized recombinant Mouse UCHLI HRP conjugated Detectio conjugated anti mouse U y One bottle containing 12 mL of HRP horseradish peroxidase 1 antibody Ready to use containing 20 mL of the chromogenic substrate tetra methylbenzidine Stop Solution e containing 20 mL of 1 N H2SO Ready to use C CY 8093 3 Version 140602 Mouse UCHL1 ELISA Kit CircuLex Uer Manual 4 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 e Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for sample preparation e Vortex mixer s of 450 540 nm e read at a single e Plate reader capable of measuring absorbance in 96 well plates at dual wa e Microplate washer optional Manual washing is possible but not preferable i Dual wavelengths of 450 550 or 450 595 nm can also be used The O b wavelength of 450 nm which will give a somewhat h
14. rker in humans for severe traumatic brain injury Crit Care Med 38 138 44 Related Products CircuLex Human UCHL1 ELISA Kit Cat CY 8092 CircuLex Human DJ 1 PARK7 ELISA Kit Cat CY 9050 CircuLex Human 14 3 3 Gamma ELISA Kit Cat CY 8082 CycLex Poly Ubiquitinated Protein ELISA Kit Cat CY 7053 CycLex Poly Ubiquitinated Protein Enrichment amp Detection Kit Cat CY 7001 CycLex Proteasone Enrichment amp Activity Assay Kit Cat CY 7002 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 cyclexzeo jp CycLex CircuLexyproducts are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products withotit prior written approval from CycLex Co Ltd To inquire about licensing for such commercialtse please contact us via email Cat CY 8093 14 Version 140602
15. y Procedure The result is summarized in the figure below Linearity 1000 900 800 E 700 E 2 600 J a 500 amp 400 S 300 z 2 5 200 100 0 0 1 2 3 4 Sample Dilution Ratio 3 C CY 8093 12 Version 140602 Mouse UCHL1 ELISA Kit CircuLex Uer Manual 4 For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Mouse UCHLI concentrations in several cell culture supernatants A Mouse UCHL1 Conc ng mL a E Balb 3T3 MEF WRI9L BaF 3 L5178Y J774 1 P3U1 C CY 8093 13 Version 140602 Mouse UCHLI ELISA Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures References Osaka H Wang YL et al 2003 Ubiquitin carboxy terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron Hum Mol Genet 12 1945 58 2 Wilkinson K D Lee K M Deshpande S Duerksen Hughes P Boss J M Pohl J 1989 The neuron specific protein PGP 9 5 is a ubiquitin carboxyl terminal hydrolase Science 246 6703672 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 Nature 395 451 452 5 Papa L Akinyi L et al 2010 Ubiquitin C terminal hydrolase is a novel bioma
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