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1. 7 Wash 4 times as same as in step 4 8 Add 100 uL of 1X HRP conjugated Streptavidin into each well cover with plate sealer or lid and incubate at room temperature ca 25 C for 20 minutes shaking at ca 300 rpm on an orbital microplate shaker 9 Wash 4 times as same as in step 4 10 Add 100 uL of Substrate Reagent into each well Avoid exposing the microtiter plate to direct sunlight Covering the wells with e g aluminum foil is recommended Return Substrate Reagent to 4 C immediately after the necessary volume is removed 11 Incubate the wells at room temperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker Appropriate incubation time may vary and incubation time can be extended up to 30 minutes if the r action temperature is below than 20 C 12 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 13 Measure absorbance in each well using a spectrophotometric plate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the plate at 450 nm if only a single wavelength can be used Wells must be read within 30 minutes 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 paper towels No2. PCSK9 LDLR in vitro Binding Assay 3 0 2 5 2 0 15 lt 1 0 0 5 0 0 0 0 5 1 1 5 2 CaCb conc mM Cat CY 8150 15 Version 120420 A ae PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Fig 6 Effect of NaCl on PCSK9 LDLR binding in vitro 40 ng mL His tagged PCSK9 wild type was used PCSK9 LDLR in vitro Binding Assay 500 NaCl conc mM Fig 7 Effect of BSA on PCSK9 LDLR binding in vitro 40 ng mL His tagged PCSK9 wild type was used PCSK9 LDLR in vitro Binding Assay 2 0 1 5 So z 1 0 0 5 0 0 0 0 5 1 1 5 2 2 5 BSA conc 96 Cat CY 8150 16 Version 120420 7 Si PCSK9 LDLR in vitro Binding Assay Kit CircuL ex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 8 Binding capability of several recombinant PCSK9s to EGF AB domain of LDL receptor In this experiment Following recombinant PCSK9 were used PCSK9 Wild Type in culture medium Cat CY R2310 PCSK9 D374Y in culture medium Cat CY R2311 PCSK9 Wild Type A53 in culture medium Cat CY R2320 PCSK9 D374Y A53 in culture medium Cat CY R2321 PCSK9 LDLR EGF AB in vitro Binding Assay 3 5 3 0 2 5 O D374Y 29 D3AYASS o a Wild type lt 1 5 Wild type A53 1 0 0 5 0 0 0 1 10 100 1 000 10 000 PCSK9 8His co
3. A Garuti R Zhao Z McDonald M Horton J D Cohen J C and Hobbs H H 2007 J Biol Chem 282 18602 18612 Cat CY 8150 18 Version 120420 7 ag PCSK9 LDLR in vitro Binding Assay Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Related Products CircuLex Mouse Rat PCSK9 ELISA Kit Cat CY 8078 CircuLex Human PCSK9 ELISA Kit Cat CY 8079 CircuLex PCSK9 LDLR in vitro Binding Assay Kit Cat CY 8150 Anti Human PCSK9 prodomain monoclonal antibody KS 3C8 Cat CY M1032 Anti Human PCSK9 monoclonal antibody KS 4H12 Cat CY M1033 PCSK9 Wild Type in culture medium Cat CY R2310 PCSK9 A 33 53 Wild Type in culture medium Cat CY R2320 PCSK9 D374Y in culture medium Cat CY R2311 PCSK9 A 33 53 D374Y in culture medium Cat CY R2321 PCSK9 Wild Type AS53 in culture medium Cat CY R2320 PCSK9 Wild Type Cat CY R2330 PCSK9 D374Y Cat CY R2331 PCSK9 R194A Cat CY R2333 LDLR EGF AB domain Cat CY R2340 LDLR EGF AB domain Myc tagged Cat CY R2341 LDLR EGF AB domain H306Y Cat CY R2342 LDLR EGF AB domain H306Y Myc tagged Cat CY R2343 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex c0 jp URL http www cyclex co jp CycLex CircuLex products are supplied for research use only CycLex CircuLex products and components thereof may not be resold modif
4. be used to screening and characterize inhibitors of PCSK9 LDLR interaction in vitro Please consider whether your xperimental condition is appropriate rawing a direct line with the results of Assa Characteristics in page 13 17 Cat CY 8150 11 Version 120420 PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX i User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting All samples should be run in duplicate when a standard assay is being performed 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 by elevated values for wells containing no sample indicat insufficient washing If all instructions in the Detailed Protocol were followed accurately such resultsdndicate a need for washer maintenance U Overall low signal may indicate that 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 reagents included in the CycLex Research Product CircuLex PCSK9 LDLR in vitro Binding Assay Kit have been tested for stability Reagents should not be used beyond the stated expiration date Cat CY 8150 12 Version 120420 PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX B Use
5. c and an 88 decrease in the risk of coronary heart disease CHD 5 In mice adenoviral overexpression of PCSK9 results in increased plasma LDL cdeyel in normal mice but not in LDLR deficient mice 7 Deletion of PCSK9 causes an increase in level of LDLR protein but not mRNA 8 These findings lead to a hypothesis that PCSK9 exertsmts role in cholesterol metabolism through posttranslational down regulation of LDLR the receptor responsible for clearing LDL c from plasma Evidence is consistent with the secreted form of PCSK9 binding directly to the LDLR and resulting in degradation of the receptor 9 10 Zhang et al 11 localized the binding site of PCSK9 in the LDLR to the first epidermal growth factor like repeat EGF A of the extracellular domain and showed that PCSK9 binding to this site is required for LDLR degradation In light of these observations and the fact that PCSK9 in the circulation may cause the degradation of hepatic EDLER in the liver PCSK9 would seem to be an attractive drug target for lowering LDLC Principle of the Assay The CycLex Research Products CircuLex PCSK9 LDLR in vitro Binding Assay Kit is a semi quantitative solid phase binding assay between recombinant His tagged PCSK9 and recombinant LDLR AB domain Plates are pre coated with a recombinant LDLR AB domain which contains binding site for PCSK9 Biotinylated anti His tag monoclonal antibody specifically reacts with recombinant His tagged PCSK9 that has been trap
6. compound or Vehicle for 20X Test compound to each well of the microplate 2 Initiate reaction by adding 10 uL of 10X His tagged PCSK9 wild type Solution to each well and mixing thoroughly Cover with plate sealer or lid and incubate at room temperature for 2 hours shaking at ca 300 rpm on an orbital microplate shaker 3 Follow the steps 4 13 of II Stand rd Assay for Drawing Dose Response Curve in page 9 Note 1 The optimal experimental conditions must be determined by the individual user Especially appropriate concentration of 10X His tagged PCSK9 wild type Solution must be determined by drawing dose responsecurve which shows OD value does not exceed plateau range in the dose response curve Note 2 To obtain an ccurate inhibition percentage of test compounds draw a dose response curve with appropriate serial dilutions of 10X His tagged PCSK9 wild type Solution in parallel with the inhibition assay of the test compounds NOTE THE ABOVE PROCEDURES ARE INTENDED ONLY AS A GUIDELINE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY DEPENDING ON THE PARAMETERS BEING INVESTIGATED NO WARRANTY OR GUARANTEE OF PERFORMANCE USING THESE PROCEDURES IS MADE OR IMPLIED Cat CY 8150 10 Version 120420 us PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results To obtain an accurate inhibition percentage of test compounds it
7. s required to draw and calculate form a dose response curve with serial dilutions of 10X His tagged sRAGE Solution in parallel with the inhibition assay of the test compounds 1 Average the duplicate readings for each Test Compound Vehicle Control and serial dilutions of 10X His tagged sRAGE Solution The absorbance value for Vehicle Control should be greater than 1 0 with a background less than 0 25 when using Reaction buffer 2 Inhibition percentage of test compounds should be calculated from the dose response curve 3 The dose response curve of this assay fits to a sigmoidal four parameter logistic equation The results of unknown samples can be calculated with any computer program having a four parameter logistic function It is important to make an appropriate mathematical adjustment to accommodate for inhibition percentage 4 Most microtiter plate readers perform automatic calculations of analyte concentration The calibration curve is constructed by plotting the absorbance Y of calibrators versus log of the known concentration X of calibrators using the four paramet r 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 Note The CycLex Research Product CirceuLex PCSK9 LDLR in vitro Binding Assay Kit has been shown to measure PCSK9 LDLR interaction in vitro The assay may
8. 7 ag PCSK9 LDLR in vitro Binding Assay Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Kit for Measuring PCSK9 LDLR interaction in vitro CircuLex PCSK9 LDLR in vitro Binding Assay Kit Cat CY 8150 Int ded NER p 1 wjfaj c E 1 Introduction cccccccceessececeesssseeeessseeeeees 2 Principle of the Assay cviccssaisacstendesedatecexivtes 2 3 Summary of Procedure ssssss 4 Materials Provided acis osntuise teas itis 5 Materials Required but not Provided 6 Precautions and Recommendations 7 Detailed Protecal decens tasti irc idet tans 8 10 Evaluation of Results ee 11 Troubleshooting eese 12 Reagent Stability simos chi tuse ee 12 Assay Characteristics 25 tine taies 13 17 TGs esner 18 Related Product 19 Intended Use The CycLex Research Product CircuLex PCSK9 LDLR in vitro Binding Assay Kit is primarily designed to screening inhibitors of PCSK9 EDLR interaction in vitro In addition this kit can be used for the rapid and sensitive evaluation of inhibitor candidates as well as characterization of PCSK9 LDLR interaction in vitromThe recombinant LDLR EGF AB domain used in this assay kit was specially designed by CycLex for keeping the correct conformation to bind PCSK9 even if immobilized on the microplate surface This t chnique allows us measuring effici
9. ently the interaction of recombinant PCSK9 to recombinant LDER AB domain in a solid phase assay system such as a conventional ELISA system Applications for this kitanclude 1 Screening inhibitors of PCSK9 LDLR interaction in vitro 2 Characterization of inhibitor candidates of PCSK9 LDLR interaction in vitro 3 Screening monoclonal antibody that inhibits PCSK9 LDLR interaction in vitro 4 Characteriz tion of PCSK9 LDLR interaction in vitro This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage Upon receipt store all components at 4 C Don t expose reagents to excessive light Cat CY 8150 1 Version 120420 us PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Introduction PCSK9 also known as neural apoptosis regulated convertase NARC 1 is a 692 residue extracellular protein expressed primarily in the kidneys liver and intestines 1 representing the 9th member of the secretory subtilase family Various genetic observations subsequently mapped PCSK9 as the third gene along with LDLR and APOB to cause autosomal dominant hypercholesterolemia ADH These studies suggested that gain of function mutations increase plasma levels of LDL c 2 6 whereas nonsense or missense loss of function mutations which interfere with folding or secretion of PCSK9 lead to a reduction of plasma levels of LDL
10. esidue from glassware Use deionized water of the highest quality Do not mix reagents from different kits The buffers and reagents used in this kit contain either sodium Kathon CG as preservatives Care should 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 assay or in areas where samples or reagents are handled Dispose of tetra methylbenzidine TMB containing solutions in compliance with local regulations CAUTION Sulfuric Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 8150 T Version 120420 7 ag PCSK9 LDLR in vitro Binding Assay Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CirceuLex PCSK9 LDLR in vitro Binding Assay 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 conditions may vary individual user should optimize the amount of Recombinant His tagged PCSK9 wild type for inhibitor screening from dose response curve Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross contamination of reagents or samples Preparation of Working Solutions 1 Prepare a working solution of Wash Buffer by addi
11. ied for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire about licensing for suchcommercial use please contact us via email Cat CY 8150 19 Version 120420
12. 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 Wash bottle or multichannel dispenser for plate washing Microcentrifuge and tubes for sample preparation Vortex mixer Microplate washer optional Manual washing is possible but not preferable Plate reader capable of measuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used The plate can also be read at a single wavelength of 450 nm which will give a somewhat higher reading Software package facilitating data generation and analysis optional 500 or 1000 mL graduated cylinder Reagent reservoirs Deionized water of the highest quality Disposable paper towels Cat CY 8150 6 Version 120420 7 ag PCSK9 LDLR in vitro Binding Assay Kit CircuLex User s Manual 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 pouch which 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 r
13. nc ng ml Cat CY 8150 17 Version 120420 us PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures References j Seidah NG Benjannet S Wickham L Marcinkiewicz J Jasmin SB Stifani S Basak A Prat A Chretien M 2003 Proc Natl Acad Sci USA 100 928 933 2 Abifadel M Varret M Rabes JP Allard D Ouguerram K Devillers M Cruaud C Benjannet S Wickham L Erlich D et al 2003 Nat Genet 34 154 156 U Leren TP 2004 Clin Genet 65 419 422 4 Allard D Amsellem S Abifadel M Trillard M Devillers M Luc G Krempf M Reznik Y Girardet JP Fredenrich A et al 2005 Hum Mutat 26 497 Nn Cohen JC Boerwinkle E Mosley TH Jr Hobbs HH 2006 N Engl J Med 354 264 1272 ON Berge KE Ose L Leren TP 2006 Arterioscler Thromb Vasc Biol 26 1094 1100 7 Maxwell KN Breslow JL 2004 Proc Natl Acad Sci USA 101 7100 7105 oo Rashid S Curtis DE Garuti R Anderson NN Bashmakov Y Ho YK Hammer RE Moon YA Horton JD 2005 Proc Natl Acad Sci USA 102 5374 5379 9 Lagace T A Curtis D E Garuti R McNutt M C Park S W Prather H B Anderson N N Ho Y K Hammer R E and Horton J D 2006 J Clin Investig 116 2995 3005 10 Cameron J Holla O L Ranheim T Kuls th M A Berge K E and Leren T P 2006 Hum Mol Genet 15 1551 1558 11 Zhang D W Lagace T
14. ng 100 mL of the 10X Wash Buffer provided to 900 mL of deionized water Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Reconstitute the Recombinant His tagged PCSK9 wild type with 1 2 mL of ddH O After dissolved by gently mixing immediately dispense in small aliquots e g 200 4L to micro centrifuge tubes and store at below 70 C to avoid repeated freezing and thawing Final concentration of recombinant His tagged PCSK9 wild type solution should be 1 ug mL which is referred as a Master solution of Recombinant His tagged PCSK9 wild type U Prepare His tagged PCSK9 wild type Solutions by 2 fold serially diluting the Master solution 1 ug mL to an appropriate concentration usually 400 1 56 ng mL only for drawing dose response curve with Reaction Buffer See the section Standard Assay for Drawing Dose Response Curve in page 9 at the time of assay 4 Prepare 1X Biotinylated anti His tag monoclonal antibody by diluting the 150X Biotinylated anti His tag monoclonal antibody solution 150 fold with Conjugate Dilution Buffer at the time of assay Nn Prepare 1X HRP conjugated Streptavidin by diluting the 150X HRP conjugated Streptavidin solution 150 fold with Conjugate Dilution Buffer at the time of assay For an inhibitor screening assay the Master solution 2 g mL should be diluted with Reaction Buffer to appropriate c ncentration which shows OD value does not exceed plateau range in d
15. oated with Recombinant LDL EGF AB domain Recombinant His tagged PCSK9 wild type One vial containing 1 2 ug of lyophilized recombinant His tagged PCSK9 wild type 150X Biotinylated anti His tag monoclonal antibody One vial containing 100 uL of 150X Biotinylated anti His tag monoclonal antibody For long term storage unused 150X Biotinylated anti His tag monoclonal antibody solution can be stored in small aliquots e g 20 pL at below 70 C to avoid repeated freezing and thawing Reaction Buffer One bottle containing 50 mL of 1X buffer used for binding reaction and dilution of Recombinant His tagged PCSK9 wild type Ready to use 150X HRP conjugated Streptavidin One vial containing 100 uL of 150X HRP conjugated Streptavidin For long term storage unused 150X HRP conjugated Streptavidin solution can be stored in small aliquots e g 20 uL at below 70 C to avoid repeated freezing and thawing Conjugate Dilution Buffer One bottle containing 30 mL of 1X buffer used for dilution of HRP conjugated Streptavidin Ready to use 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing 2 Tween 20 Substrate Reagent One bottle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mof 1 N H2SOx Ready to use Cat CY 8150 5 Version 120420 ag PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX User s Manual For Research Use Only Not for use
16. ose response curve Prepare appropriate volume for your assay Discard any unused Recombinant His tagged sRAGE Solutions 1X Biotinylated anti His tag monoclonal antibody and 1X HRP conjugated Streptavidin after diluted Cat CY 8150 8 Version 120420 us PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay for Drawing Dose Response Curve 1 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 Prepare 2 fold serially diluted His tagged PCSK9 wild type Solutions 100 1 56 ng mL and Reaction Buffer only 3 Pipette 100 uL of the His tagged PCSK9 wild type Solutions 100 1 56 ng mL and Reaction Buffer only in duplicates into the wells Cover with plate sealer or lid and incubate atoom temperature ca 25 C for 2 hours shaking at ca 300 rpm on an orbital microplate shaker 4 Wash 4 times by filling each well with Wash Buffer 350 uL using a squirt bottle multi channel pipette manifold dispenser or microplate washer Remove residual Wash Buffer by gentle tapping or aspiration 6 Add 100 uL of 1X Biotinylated anti His tag monoclonal antibody into each well cover with plate sealer or lid and incubate at room temperature ca 25 C for T hour shaking at ca 300 rpm on an orbital microplate shaker
17. pedgwith recombinant LDLR AB domain immobilized on the microplate surface The CircuLex PCSK9 LDLR in vitro Binding Assay Kit may be used to characterize PCSK9 LDLR interaction as well as to screening inhibitors of PCSK9 LDLR interaction in vitro To perform the test the recombinant His tagged PCSK9 is diluted in Reaction Buffer pipetted into the wells and allowed to bind recombinant LDLR AB domain immobilized on the wells After wash the plate the amount of bound recombinant His tagged PCSK9 is measured by binding it with biotinylated anti His tag monoclonal antibody followed by binding with horseradish peroxidase conjugated Streptavidin which then catalyzes the conversion of the chromogenic substrate tetra methylbenzidine TMB from a colorless solution to a blue solution or yellow after the addition of stopping reagent The color is quantitated by spectrophotometry and reflects the relative amount of recombinant His tagged PCSK that binds to recombinant LDLR AB on the microplate For screening inhibitors of PCSK9 LDLR interaction in vitro test compounds or the inhibitor candidates including monoclonal antibody is added to appropriate amount of His tagged PCSK9 in the wells that have been coated with recombinant LDLR AB domain in a similar fashion as described above followed by evaluation of inhibitory effect on PCSK9 LDLR interaction by measuring the amount of His tagged PCSK9 on the wells Cat CY 8150 2 Version 120420 A s PCSK9 LDLR in
18. r s Manual For Research Use Only Not for use in diagnostic procedures Assay Characteristics Fig 1 Typical dose dependent curve of Recombinant His tagged PCSK9 wild type pH 6 5 PCSK9 LDLR in vitro Binding Assay 3 5 A450 0 50 100 150 PCSK9 8His conc ng ml 200 Fig 2 Time course of PCSK9 LDLR binding assay in vitro 80 ng mL His tagged PCSK9 wild type was used PH 7 2 PCSK9 LDLR in vitro Binding Assay 0 60 120 180 240 Reaction Time mm Cat CY 8150 13 Version 120420 T PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of pH on PCSK9 LDLR binding in vitro 80 ng mL His tagged PCSK9 wild type was used PCSK9 LDLR in vitro Binding Assay Fig 4 Effect of DMSO on PCSK9 LDLR binding in vitro PCSK9 LDLR in vitro Binding Assay 3 0 2 5 2 0 z 1 5 1 0 0 5 0 0 0 1 2 3 4 5 DMSO conc Cat CY 8150 14 Version 120420 us PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Fig 5 Effect of calcium ion on PCSK9 LDLR binding in vitro 40 ng mL His tagged PCSK9 wild type was used PCSK9 LDLR in vitro Binding Assay 200 300 CaCb conc uM
19. te 2 Reliable signals are obtained when either O D values do not exceed 0 25 units for the blank Reaction Buffer only or 3 0 units for the 100 ng of His tagged PCSK9 wild type Solution Cat CY 8150 9 Version 120420 PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX i User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay for Inhibitor Screening Special considerations when screening inhibitors of PCSK9 LDLR interaction In order to estimate the inhibitory effect on PCSK9 LDLR interaction in the test compounds correctly it is necessary to conduct the control experiment of Vehicle Control at least once for every experiment in addition to Test Compound as indicated in the following table When test compounds cause an inhibitory effect on PCSK9 LDLR interaction the level of A450 is weakened as compared with Vehicle Control For inhibitor screening Assay Reagents Test Compound Vehicle Control Reaction buffer 85 uL 85 uL 20X Test Compound s 5 uL Vehicle for 20X Test Compound 5uL 10X His tagged PCSK9 wildtypeSolution Dub en luL 10x concentrated solution of the His tagged PCSK9 wild type Solution which shows OD value does not exceed plateau range in dose response curve Usually 500 750 ng mL see below See the section I Preparation of Working Solutions in pages 1 Following the above table add the Reaction buffer and 20X Test
20. vitro Binding Assay Kit Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Schematic Representation of the CircuLex PCSK9 LDLR in vitro Binding Assay Kit Principle M wash wash LDLR EGF AB Y Biotinylated anti His tag mAb X His tagged PCSK9 ad HRP conjugated Streptavidine 3 TMB Cat CY 8150 3 Version 120420 7 ag PCSK9 LDLR in vitro Binding Assay Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of recombinant His tagged PCSK9 to the wells Y Incubate for 2 hrs at room temp Wash the wells Add 100 pL of biotinylated Anti His tag monoclonal Antibody Y Incubate for 1 hr at room temp Wash the wells Add 100 pL of HRP conjugated Streptavidin Y Incubate for 20 mih at room temp Wash the wells Y Add 100 uL of Substrate Reagent Y Add 100 pL of Stop Solution Y Measure absorbance at 450 nm Cat CY 8150 4 Version 120420 us PCSK9 LDLR in vitro Binding Assay Kit Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided All samples should be assayed in duplicate The following components are supplied and are sufficient for the one 96 well microtiter plate kit Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are c
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