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1. Nasu H Hibi N Tsukada Y Ohkawa K fujimuro M Sawada H and Yokosawa H Eur J Biochem 233 42 47 1995 amp Related Products CycLex Poly Ubiquitinated Protein Enrichment Detection Kit Cat CY 7001 CycLex Proteasome Enrichment amp Activity Assay Kit Cat CY 7002 CycLex Poly Ubiquitinated Protein ELISAMGit Cat CY 7053 PRODUCED BY CycLex Co Ltd N 1063 103 ren Ina Nagano 396 00 Japan Fax 81 265 e mail info CycLAdCircuLex products are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial pro ycts without prior written approval from CycLex Co Ltd To inquire about licensing for S commercial use please contact us via email 2 Roa CY 7053 14 Version 150825 o c2. Z CY 7053 9 Version 150825 Kor CY 7053 10 Version 150825 ge P 4 r ycLex User s Manual For Research Use Only Not for use in diagnostic procedures A S Poly Ubiquitinated Protein ELISA Kit S 7 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an q l microplate shaker 8 Wash 4 times by filling each well with Wash Buffer 350 uL using a squirt bottle mi hannel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent Avoid exposing the microtiter plate to direct ght covering the plate with e g aluminum foil is recommended Return Substrate A to 4 C i iately after the necessary volume is removed rd 10 Incubate the plate at room temperature ca 25 C for 10 20 minutes sh at ca 300 rpm on an orbital microplate shaker The incubation time may be extended up t minutes if the reaction temperature is below than 20 C bo N 11 Add 100 uL of Stop Solution to each well in the same order a previously added Substrate Reagent A eo 12 Measure absorbance in each well using a eb E lt a aes reader at dual wavelengths at 450 nm if only a single wavelength can be used Wells ust be read within 30 minutes of adding of 450 540 nm Dual wavelengths of 450 550 or 450 595 an also be used Read the microplate the Stop Solution 7 Note 1 Complete removal of liquid at each step is essgntial to good performance After the last wa
3. Buffer 350 uL using a sgi Bue multi channel pipette manifold dispenser or microplate washer 6 Add 100 uL of HRP conjugated Detection Antibody into each well xs N 7 Incubate the plate at room temperature ca 25 C for 1 hour shah at ca 300 rpm on an orbital microplate shaker 8 Wash 4 times by filling each well with Wash Buffer 350 using a squirt bottle multi channel pipette manifold dispenser or microplate washer lt 9 Add 100 uL of Substrate Reagent Avoid exposing ing Pcroriter plate to direct sunlight covering the plate with e g aluminum foil is recommended Retyfn Substrate A to 4 C immediately after the necessary volume is removed eA 10 Incubate the plate at room temperature ca 2 for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation tie may be extended up to 30 minutes if the reaction temperature is below than 20 C 11 Add 100 uL of Stop Solution to eagen in the same order as the previously added Substrate Reagent 74 x 12 Measure absorbance in each well sing a spectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelen gO of 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single KA ength can be used Wells must be read within 30 minutes of adding the Stop Solution Assay Procedure 2 a cultured in 96 wells culture plate A Treatment of cousin compounds 1 Plate adherent oGths or non adher
4. Protocol 4 The CycLex Research Product CycLex Poly Ubiquitinated Protein ELISA Kit is pro 2d with removable strips of wells so the assay can be carried out on separate occasions using only thesaumber of strips required for the particular determination Since experimental conditions may vary aliquot of the Poly Ubiquitinated Protein Standard within the kit should be included in each assay g a calibrator Disposable pipette tips and reagent troughs should be used for all liquid iver to avoid cross contamination of reagents or samples Y Q Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay reagents are supplied ready to use with the exception of 10X Wash Buffer Cell Extraction B and Poly Ubiquitinated Protein Standard gt Prepare a working solution of Wash Buffer by adding 100 mL vere Wash Buffer to 900 mL of deionized distilled water Mix well Store at 4 C for two weeks 0 C for long term storage 2 Prepare a working solution of Cell Extraction Buffer by addi 50 uL of Protease inhibitor cocktail Sigma Cat P 2714 to 5 mL of Cell Extraction Buffer Nix well 3 Prepare HRP conjugated Detection Antibody by dire the 20X HRP conjugated Detection Antibody 20 fold with Conjugate Dilution Buffer at th time of assay Prepare appropriate volume for your assay card any unused HRP conjugated Detection Antibody after diluted A 4 Reconstitute Poly Ubiquitinated Protein S
5. S Poly Ubiquitinated Protein ELISA Kit Pa P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures gt ELISA Kit for Measuring Poly Ubiquitinated Protein amp CycLex Poly Ubiquitinated Protej ELISA Kit Cat CY 7053 2 ARV amp Intended US xcovcsazasnceasecenevieccccaxteeaenkacsend 1 aS SOAD E eee eeneerets 1 A ntroduction s ssseeeeeeeeeeeseeseseesrsssresesessrsses 2 RG Principle of the Assay 2 3 amp Materials Provided s sissressiscsaasessansoxtaieeniacs 4 Yon Materials Required but not Provided 4 Precautions and Recommendations 5 amp Detailed Protocol ssssssssssssssssssseeeesssneeees 6 10 K ALCMLAUIONS 352s cavactwasedacesansgrearncont shommanes 11 K Measurement Range 11 Troubleshooting 1 Reagent Stability cccccccesssesesseeseeneeeeen IN Assay Characteristics ceeeceseeeeereeees Example of Test Results eee amp 3 Referente Sisaria anas O 14 Related Products is icsscccsesisevsaacieestens Cy 14 Intended Use G The CycLex Research Product aex Poly Ubiquitinated Protein ELISA Kit is designed to detect and quantify the level of total Ob iquitinated proteins in cell lysate Since the amino acid sequence of ubiquitin is well conserved wp mammal this ELISA kit can be used for any mammalian cells This assay is intended for the detecf amp yon of poly ubiquitinated proteins in cell lysate a This assay kit i
6. cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Materials Provided 3 All samples and standards should be assayed in duplicate The following components are uta and are sufficient for the one 96 well microplate kit RY Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in yfoil zip lock bag with a desiccant pack Wells are coated with anti poly ubiquitinated protein mongrel antibody as a capture antibody a 10X Wash Buffer One 100 mL bottle of 10X buffer containing 2 Tween 20 Mag Cell Extraction Buffer One bottle containing 20 mL of 1X buffer Rd Dilution Buffer One bottle containing 50 mL each of 1X buffer use fa andard and sample dilution Ready to use wy Poly Ubiquitinated Protein Standard One vial containing 150 fs of lyophilized poly ubiquitinated protein 20X HRP conjugated Detection Antibody One vial co yaining 0 6 mL o HRP horseradish peroxidase conjugated anti poly ubiquitin monocl antibody Conjugate Dilution Buffer One bottle containing 12 of Conjugate Dilution Buffer Substrate Reagent 20 mL of the chromogenic apenas tetra methylbenzidine TMB Ready to use Stop Solution One bottle supplied ready to NS iting 20 mL of 1 N H2SOa R Materials Required but no e Orbital microplate shaker 74 e Protease inhibitor cocktail gaen Cat P 2714 reconstituted according to manufacturer s guideline Add 250 uL per 5 ell Ext
7. can enter mammalian cells and inhibit de ation of proteins by the ubiquitin proteasome pathway One group of such inhibitors is YN aldehydes e g MG132 which reversibly binds to active sites and inhibits cleavage of hydrophobi acidic substrates A more specific inhibitor is the naturally occurring bacterial compound lactaysth hich covalently modifies threonine residues in the proteasome s active site and does not seem fect any other known protease Such agents can inhibit protein degradation and major histocomg bility class I antigen presentation in a variety of mammalian cells and have been widely use Co probe the physiological function of the ubiquitin proteasome pathway Se Principle of the Assay Xs The CycLex Research CycLex Poly Ubiquitfpated Protein ELISA Kit is a solid phase sandwich ELISA A monoclonal antibody specific polygbiquitinated protein has been coated onto the wells of the microtiter strips provided Samples inclu j a standard containing poly ubiquitinated protein control specimens and unknowns are pipetted i hese wells During the first incubation poly ubiquitinated protein binds to the capture antibody of e well After washing HRP conjugated mouse monoclonal antibody specific poly ubiquitinated ptein as a detection antibody is added to the wells During the second incubation this antibody segs as a detector by binding to the immobilized poly ubiquitinated protein at captured during the firsacubation After removal of exce
8. e from glassware e Use deionized water of the highest quality RU S Y A N e Do not mix reagents from different kits amp The buffers and reagents in this kit may contain preservatives ier chemicals Care should be taken to avoid direct contact with these reagents Do not mouth pipette or ingest any of the reagents RZ e Do not smoke eat or drink when performing the say or in areas where samples or reagents are handled A e Dispose of tetra methylbenzidine TMB contei g solutions in compliance with local regulations e Avoid contact with the acidic Stop Soluig Substrate Solution which contains hydrogen peroxide e Wear gloves and eye protection whet Fandting immunodiagnostic materials and samples of human origin and these reagents In case ontact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and se gmedical attention when necessary Biological samples may Pacer ssaiel with infectious agents Do not ingest expose to open wounds or breathe aerosdls Wear protective gloves and dispose of biological samples properly handli p Solution O nw a Qe amp eo Roa CY 7053 5 Version 150825 e CAUTION handia id is a strong acid Wear disposable gloves and eye protection when o AO Poly Ubiquitinated Protein ELISA Kit e cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures A Q O Q 4 mma o Detailed
9. ein 4 Shake the 96 wells culture plate at ca 300 rpm on an orbital govt shaker at 4 C for 1 hr Centrifuge the 96 wells culture plate at 3 000 rpm at 4 CGar 15 min 6 Carefully transfer the lysates to new a 96 wells micrepjate The clear lysates in the 96 wells microplate arene dy for assay These cell lysates can be stored at below 70 C Avoid multiple freeze thaw oO C ELISA lt Remove the appropriate number of ee wells from the foil pouch and place them into the well holder Return any unused wells to A oil pouch refold seal with tape and store at 4 C 2 Dilute the lysates from 96 wel Alte 1 50 with Dilution Buffer e g 5 uL cell lysate 245 uL Dilution Buffer Lysates prepared in Cell Bxtraction Buffer must be diluted 1 50 or greater in Dilution Buffer While a 1 50 sample dilution has been found to be satisfactory higher dilutions such as 1 100 or 1 200 may be optimal TA Yrion chosen should be optimized for each experimental system 3 Pipette 100 uL oSAndara Solutions Std1 Std7 Blank and diluted lysates in duplicates into the appropriate welgy 4 Incubate theBjate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital micropla aker 5 Wagyrines by filling each well with Wash Buffer 350 uL using a squirt bottle multi channel pipette manifold dispenser or microplate washer og 100 uL of HRP conjugated Detection Antibody into each well rA
10. ent cells in a 96 wells culture plate at 50 70 confluency 2 Incubate th wells culture plate at 37 C over night in CO2 incubator 3 Add angapprate amount of test compounds to each well 4 nye the 96 wells culture plate at 37 C for appropriate time ack Extraction c pi 3 Add 50 pL of Cell Extraction Buffer to each well AY n N Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures A S Poly Ubiquitinated Protein ELISA Kit S Note This protocol has been successfully applied to several cell lines Users should optimize th Q extraction procedure for their own applications AN Remove the culture medium and wash the cells twice with 200 uL of cold PBS using a c uge for microplate 2 Remove PBS completely Tap the 96 wells culture plate on the paper towel in ca the adherent cells Take PBS away carefully using multi channel pipette in case of the non ad t cells At this point the 96 wells culture plate can be frozen at below 70 C and lyse at a later NU The volume of Cell Extraction Buffer depends on the cell line the ce 1 faber in cell pellet and the amount of poly ubiquitinated protein For example 1 2 x 104 MCF 7 ls can be extracted in 50 uL of Cell Extraction Buffer Under these conditions use of 0 5 2 uL e clarified cell lysate diluted to a volume of 100 uL well in Dilution Buffer is sufficient for Qe detection of poly ubiquitinated prot
11. in ELISA kit MCE7 cell lysates after10uM MG 132 treatment N 3 0 p 2 W O D N Y Ni G 0 4 8 12 16 20 24 28 32 Treatment time hr o gL eA Roa CY 7053 13 Version 150825 amp lt C A y Poly Ubiquitinated Protein ELISA Kit Pe r ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 References 3 AYN oO NDU 9 Goldberg A L Stein R Adams J Chem Biol 2 503 508 1995 aS Coux O Tanaka K Goldberg A L Annu Rev Biochem 65 801 847 1996 3 King R W Deshaies R J Peters J M Kirschner M W Science 274 1652 1659 1996s Chau V Tobias J W Bachmair A Marriott D Ecker D J Gonda D K VarshavgRy A Science 243 1576 1583 1989 Hochstrasser M Curr Opin Cell Biol 7 215 223 1995 Y Ciechanover A Cell 79 13 21 1994 Q Jentsch S and Schlenker S Cell 82 881 884 1995 RU Rock K L Gramm C Rothstein L Clark K Stein R Dick L Be and Goldberg A L Cell 78 761 771 1994 Jensen T J Loo M A Pind S Williams D B Goldberg A Lai Riordan J R Cell 83 129 135 1994 Y 10 Ward C L Omura S and Kopito R R Cell 83 121 127 1995 11 Fenteany G Standaert R F Lane W S Choi S Corey E J Qrd Schreiber S L Science 268 726 731 1995 12 Fujimuro M Sawada H and Yokosawa H FEBS Lett 373 20 1994 13 Takada K
12. linearized by using log log paper a eee analysis may be applied to the log transformation To determine the poly ubiquiffgated protein concentration of each sample first find the absorbance value on the y axis and exten orizontal line to the standard curve At the point of intersection extend a vertical line to the is and read the corresponding poly ubiquitinated protein concentration If the samples hav y een diluted the concentration read from the standard curve must be multiplied by the dilution faetor 1 The dose response curve of this assay fits best to a sigmoidal ESA logistic equation The results of unknown samples can be calculated with any computer progfam having a 5 parameter logistic function It is important to make an appropriate mathematical ANustment to accommodate for the dilution factor wy 2 Most microtiter plate readers perform automatic calculations of flyte concentration The calibration curve is constructed by plotting the absorbance Y of Moravors versus log of the known concentration X of calibrators using the four parameter function Alternatively the logit log function can be used to linearize the calibration curve i logit of absorbance Y is plotted versus log of the known concentration X of calibrators Measurement Range O The measurement range is 0 156 units mL to 1Qunits mL Any sample reading higher than the highest standard should be diluted with Dilution Buff igher dilution and re assayed Dilutio
13. n factors need to be taken into consideration in calculating thg poly ubiquitinated protein concentration Troubleshooting Pi 1 The Poly Ubiquitinated Protein Stard should be run in duplicate using the protocol described in the Detailed Protocol Incubatigfdimes or temperatures significantly different from those specified may give erroneous results Q 2 Poor duplicates T A by elevated values for wells containing no sample indicate insufficient washing If all instructiogis in the Detailed Protocol were followed accurately such results indicate a need for washer main e 3 Overall low signday indicate that desiccation of the plate has occurred between the final wash and addition of eure Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash 7 Reago Stability All gr reagents included in the CycLex Research Product CycLex Poly Ubiquitinated Protein ELISA Kit have been tested for stability Reagents should not be used beyond the stated expiration date Up ceipt kit reagents should be stored at 4 C except the reconstituted Poly Ubiquitinated Protein S d must be stored at below 70 C Coated assay plates should be stored in the original foil bag ed by the zip lock and containing a desiccant pack kA at CY 7053 11 Version 150825 o AO A S Poly Ubiquitinated Protein ELISA Kit Pe oe ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 As
14. on Buffer is sufficient for the detection of poly ubiquitinated proven 5 Transfer the lysate lagutefocentrfuge tubes and centrifuge at 15 000 rpm for 10 minutes at 4 C 6 Aliquot the clear eXtract to clean microcentrifuge tubes These lysates are ready for assay The cell lysate can be yea at below 70 C Avoid multiple freeze thaw cycles O C ELISA Q 1 Rem Ohe appropriate number of microtiter wells from the foil pouch and place them into the well holdeg Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 rate lysates 1 500 with Dilution Buffer e g 1 uL cell extract 499 uL Dilution Buffer Q Lysates prepared in Cell Extraction Buffer must be diluted 1 200 or greater in Dilution Buffer While Xi ate CY 7053 T Version 150825 o c Kor CY 7053 8 Version 150825 ee Pa P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures A S Poly Ubiquitinated Protein ELISA Kit S a 1 200 sample dilution has been found to be satisfactory higher dilutions such as 1 500 or 1 o may be optimal The dilution chosen should be optimized for each experimental system 3 Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted lysates in duplic Anto the appropriate wells 4 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300 on an orbital microplate shaker 5 Wash 4 times by filling each well with Wash
15. raction Buffer e Pipettors 2 20 uL 20 200 d 200 1000 uL precision pipettors with disposable tips e Multi channel Pipettors Q250 uL precision multi channel pipettor with disposable tips e Microcentrifuge and tuBes for sample preparation e Centrifuge and buckets microplate e Vortex mixer e Microplate wash ptional Manual washing is possible but not preferable e Plate reader capable of measuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavgl ngths of 450 550 or 450 595 nm can also be used The plate can also be read at a single wave h of 450 nm which will give a somewhat higher reading Software age facilitating data generation and analysis optional 500 or 1 graduated cylinder Reag servoirs Deiofiged water of the highest quality Disgpsable paper towels e Roa CY 7053 4 Version 150825 o AO S Poly Ubiquitinated Protein ELISA Kit 9 ycLex User s Manual 3 For Research Use Only Not for use in diagnostic procedures 4 Precautions and Recommendations 3 e Allow all the components to come to room temperature before use we All microplate strips that are not immediately required should be returned to the zip lock Na which must be carefully resealed to avoid moisture absorption g e Do not use kit components beyond the indicated kit expiration date Yon e Use only the microtiter wells provided with the kit e Rinse all detergent residu
16. s for red use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt re all components at 4 C e Don t expo agents to excessive light 2 Roa CY 7053 1 Version 150825 o c Poly Ubiquitinated Protein ELISA Kit P 4 r ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Introduction 3 contribute to controlling the intracellular levels of a variety of short lived proteins in ition to degrading abnormal proteins in the cytosol and nucleus Protein substrates are m with a poly ubiquitin chain and then degraded to peptides and free ubiquitin by a large multicagty tic complex The ubiquitin proteasome pathway is the principle pathway of proteolysis in eukaryotic a may d the proteasome which exists within all eukaryotic cells Numerous examples of regul proteins have been found to undergo ubiquitin dependent proteolysis Protein substrates of the ubiquitin proteasome pathway include a number of cell latory molecules such as cyclins the Myc oncogene protein and p53 and the regulated degradation of these molecules has been linked to the control of cell proliferation and cell cycle progresin By controlling the intracellular levels of such proteins the activity of the ubiquitin proteas k pathway might also be linked to apoptosis In a recent decade a variety of reversible and irreversible inhibitors o 20S proteasome have been identified that
17. say Characteristics 3 1 Sensitivity Ae The limit of detection defined as such a concentration of poly ubiquitinated pron giving absorbance higher than mean absorbance of blank plus three standard deviations of the rbance of blank A blank 3 SD blank is better than 0 08 units mL of sample amp Dilution Buffer is pipetted into blank wells Typical standard curve Standard Curve of Poly Ubiquitinated Protein WwW 2 5 p gt 2 0 F 1 5 f N wt lt 1 0 f 0 5 F 0 0 0 2 4 6 8 10 Poly Ubiquitinated Protein U mL 2 Specificity N The antibodies in ycLex Poly Ubiquitinated Protein ELISA Kit are highly specific of poly ubiquitinated pyefeen with no detectable reactivity to monomer ubiquitin and mono ubiquitinated proteins 3 Linearity To assess tt linearity of the assay samples containing and or spiked with high concentrations of poly ubiquitfgated protein were serially diluted with the Dilution Buffer to produce samples with values within the amic range of the assay Q S 72 Roa CY 7053 12 Version 150825 o AO ww S Poly Ubiquitinated Protein ELISA Kit Pa ca ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Example of Test Results 3 Fig Breast cancer cell line MCF 7 cells were treated with 10 uM MG132 for indicated time WN sates of the MCF 7 cells from the indicated time were measured by the CycLex Poly U itinated Prote
18. sh remove any remaining Wash Buffer by aging or decanting Invert the plate and blot it against clean paper towels Note 2 Reliable standard curves are obtained When either O D values do not exceed 0 25 units for the blank zero concentration or 2 5 unitg for the highest standard concentration The plate should be monitored at 5 minute interval approximately 30 minutes Note 3 If the microplate reader is not Sable of reading absorbance greater than the absorbance of the highest standard perform a snd reading at 405 nm A new standard curve constructed using the values measured at 40 Gh is used to determine poly ubiquitinated protein concentration of off scale samples The oo at 405 nm should not replace the on scale readings at 450 nm NOTE THE ABOVE phOCEDURES ARE INTENDED ONLY AS A GUIDELINE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY DEPENDING ON THE PARAMETE ING INVESTIGATED AND MUST BE DETERMINED BY THE INDIVIDU ER NO WARRANTY OR GUARANTEE OF PERFORMANCE USING THESE P EDURES IS MADE OR IMPLIED F amp gt o G oe Poly Ubiquitinated Protein ELISA Kit P 4 cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Calculations 3 Average the duplicate readings for each standard control and sample and subtract the a Zero standard optical density Plot the optical density for the standards versus the concentr of the standards and draw the best curve The data can be
19. ss detection antibody followed by which then catalyzes the conv n of the chromogenic substrate tetra methylbenzidine TMB from a colorless solution to a MRTN or yellow after the addition of stopping reagent The color is quantitated by spectrophotemetry and reflects the relative amount of poly ubiquitinated protein present in the original specimens oO B O RS Q e Roa CY 7053 2 Version 150825 o AO L S Poly Ubiquitinated Protein ELISA Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures gt Summary of Procedure l Culture cells in culture flask or dish at 50 70 confluency 4 Incubate O N at 37 C in a CO2 incubator RY Add appropriate amount of the drug for induction of ubiquitination amp 4 Incubate appropriate time at 37 C in a CO2 dion Harvest the cells by scraping or centrifugation Q 1 gt Make cell extract by adding extraction buffer and centrifugation mS gt Add 100 uL of diluted cell lysate to the wells 2 4 Incubate for 1 hour at room mp Wash the wells i go Add 100 uL of HRP conjugated Antibody HRRG njugated anti poly ubiquitin mAb 4 Incubate for Ihor at room temp Wash the wells SS O Add 100 uL of Substrate Reagent 4 sama Add 100 uL of Stop Solution amp G t o Measure absorbance at nm Qe gL eA Roa CY 7053 3 Version 150825 o c A S Poly Ubiquitinated Protein ELISA Kit e
20. tandard with 1 mL of Dilution Buffer The concentration of the poly ubiquitinated progin in vial should be 150 units mL which is referred as a Master Standard Prepare Standard Solutions as follows Use the Master Standard to p dguce a dilution series below Mix each tube thoroughly before the next transfer The 10 unit L standard Std 1 serves as the highest standard The Dilution Buffer serves as the zero dud Blank Q of Standard Dilution Buffer Concentration Std 1 40 ULN Master Standard 560 uL 10 units mL Std 2 30 f Std 1 10 units mL 300 uL 5 units mL Std 3 L of Std 2 5 units mL 300 uL 2 5 units mL Std 4 0 uL of Std 3 2 5 units mL 300 uL 1 25 units mL Std 5 300 uL of Std 4 1 25 units mL 300 uL 0 625 units mL Std 300 uL of Std 5 0 625 units mL 300 uL 0 313 units mL 300 uL of Std 6 0 313 units mL 300 uL 0 156 units mL gam 300 uL 0 units mL Nof s 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 S 70 C immediately Avoid multiple freeze and thaw cycles Roa CY 7053 6 Version 150825 o AO S Poly Ubiquitinated Protein ELISA Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures S Assay Procedure 1 For cells cultured in culture dish or flask A Treatment of Cells
21. with compounds S 1 Plate adherent cells or non adherent cells in culture dish or flasks at 50 70 confluency KS 2 Incubate the culture dish or flasks at 37 C over night in CO2 incubator N 3 Add appropriate amount of test compounds to each dish or flask 2 4 Incubate the culture flasks at 37 C for appropriate time B Cell Extraction A D gt Note This protocol has been successfully applied to several cadies Users should optimize the cell extraction procedure for their own applications y 1 Collect cells in PBS by centrifugation non adherent cells raping from culture flasks adherent cells 2 Wash cells twice with cold PBS 3 Remove and discard the supernatant and collec Wel pellet At this point the cell pellet can be frozen at below 70 C and lysed at a later date x 4 Lyse the cell pellet in 0 5 mL of Cell Exton Buffer for 30 minutes on ice with vortexing at 10 minute intervals amp To get a rough idea you could adju e cell concentration to around 2 x 10 cells mL Resulting protein concentration of the cell lystleg Should be 2 4 mg mL using this Cell Extraction Buffer The volume of Cell Extraction Baffer depends on the cell line the cell number in cell pellet and the amount of poly ubiquitinated Ptein For example 1 x 10 MCF 7 cells can be extracted in 0 5 mL of Cell Extraction Buffers der these conditions use of 0 1 0 5 uL of the clarified cell lysate diluted to a volume 100 uL well in Diluti
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