CircuLex PCSK9-cIAP1 in vitro Binding Assay Kit
Contents
1. For inhibitor screening Assay Reagents Test Compound Vehicle Control Reaction buffer 85 uL 85 uL 20X Test Compound s 5yuL Vehicle for 20X Test Compound 5uL 10X His tagged PCSK9 wild type Solution l10uL m i Ay eee 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 5002 50 ng mL see below See the section I Preparation of Working Solutions in page 7 Following the above table add the Reaction buffer and 20X Test 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 11 of Standard Assay for Drawing Dose Response Curve in page 8 Note 1 The optimal experimental condition must be determined by the individual user Especially appropriate concentfatiomlof 10X His tagged PCSK9 wild type Solution must be determined by drawing dose reSponse curve which shows OD value does not exceed plateau range in the dose response Curve Note 2 To obtain an accurat inhibition percentage of test compounds draw a dose response curve with appropriate serial dilutions of 10X His tagged P2. tions in compliance with local regulations CAUTION Sulfuric Acid is a strong acid far sposable gloves and eye protection when handling Stop Solution Y 8152 6 Version 150127 r mp PCSK9 cIAP1 in vitro Binding Assay Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex PCSK9 cIAP1 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 l Prepare a working solution of Wash Buffer by adding 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 0 6mIz of ddH2O After dissolved by gently mixing immediately dispense in small aliquots e g 200 uL to micro centrifuge tubes and store at below 70 C to avoid repeated freeZing atnd thawing The final concentration of recombinant His tagged PCSK9 wild type solution should be 1 5
3. Horton JD 2005 Proc Natl Acad Sci USA 102 5374 5379 9 Xu W Liu L Hornby D 2012 Molecules 2012 17 12086212101 Related Products CircuLex PCSK9 LDLR in vitro Binding AsSay Kit Cat CY 8150 CircuLex PCSK9 cIAPI in vitro Binding Assay Kit Cat CY 8152 CircuLex Mouse Rat PCSK9 ELISA Kit Cat CY 8078 CircuLex Human PCSK9 ELISA Kit Cat sCY 8079 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 A53 in culture medium Cat CY R2320 PCSK9 Wild Type Cat CY R2330 PCSK9 D374Y Cat CY R2331 PCSK9 R194A Cat Y 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 Cat CY 8152 17 Versions 150127 n m PCSK9 cIAP1 in vitro Binding Assay Kit C rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures A 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 cyclex co jp CycLex CircuLex products are supplied for research use only Cy
4. of PCSK9 lead to a reduction of plasma levels of LDL 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 c level 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 exerts its role in cholesterol metabolism throughgposttranslational down regulation of LDLR the receptor responsible for clearing LDL c from plasma By shotgun proteomic analysis Weiming Xu et al identified cellular inhibitoryof apoptosis protein 1 cIAP1 as a binding partner of PCSK9 9 They revealed that cIAP Lamight regulate the activities of PCSK9 by two ways promoting PCSK9 maturation from the proprotein form to the mature form and leading to ubiquitination of PCSK9 to lysosomal d graddtion The following compelling evidences support this conclusion Knockdown of c IAP impairs PCSK9 processing and autocatalytic cleavage In c IAP1 null MEFs there is a dramatic decr ase in secreted mature PCSK9 protein while in the wild type MEF cells proprotein form of the PCSK9 was properly processed and secreted into cell supernatants Principle of the Assay The CycLex Research Products CircuLex PCSK9 cIAPI in vitro Binding Assay Kit is a semi quantitative solid phase binding assay between recombinant His tagged PCSK9 and recombinant human cIAPI Plates are pre coated w
5. CSK9 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 8152 9 Version 150127 PCSK9 cIAP1 in vitro Binding Assay Kit C ircuLex y 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 s required to draw and calculate form a dose response curve with serial dilutions of 10X His tagged PCSK9 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 PCSK9 Solution The absorbance value for VehiclegGontrok 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 CUtye W The dose response curve of this assay fits to a sigmoidal four parameter logistie equation The results of unknown samples can be calculated with any computer progfam having a four parameter logistic function It is important to make an appropriate mathematical adjustment to accommodate for inhibition percentage 4 Most microti
6. bance in 96 well plates at d elengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also b plate can also be read at a single wavelength of 450 nm which will give a somewhat ading Software package facilitating data generation and analysis o 500 or 1000 mL graduated cylinder Reagent reservoirs Deionized water of the highest quality Disposable paper towels Y 8152 5 Version 150127 PCSK9 cIAP1 in vitro Binding Assay Kit CircuLex Users Maal For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations 4 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 residue from glassware Use deionized water of the highest quality Qy Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or oth icals 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 are ere samples or reagents are handled e Dispose of tetra methylbenzidine TMB containing sol
7. cLex CircuLex products and components thereof may not be resold modified for resale or used to manuf ommercial products without prior written approval from CycLex Co Ltd To inquire a ensing for such commercial use please contact us via email G Eo Y 8152 18 Version 150127
8. cate a need for washer maintenance 3 Overall low signal may indicate that desiccation of the plate has occurred between th ash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash Reagent Stabilit All of the reagents included in the CycLex Research Product CircuLex CIAPI in vitro Binding Assay Kit have been tested for stability Reagents should n beyond the stated expiration date Y 8152 11 Version 150127 CircuLex Assay Characteristics For Research Use Only Not for use in diagnostic procedures PCSK9 cIAP1 in vitro Binding Assay Kit User s Manual Fig 1 Typical dose dependent curve of Recombinant His tagged PCSK9 wild type pH 5 0 PCSK9 cIAPI in vitro Binding Assay Dose Depwndency 40 60 100 120 His tagged PCSK wild type conc ng ml Fig 2 Effect of pH on PCSK9 cIAPI binding in vitro 150 ng mL His tagged PCSK9 wild type was used Cat CY 8152 3 0 2 5 PCSK9 cIAP1 Binding Assay pH 12 Version 150127 PCSK9 cIAP1 in vitro Binding Assay Kit C ircuLex i User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of DMSO on PCSKO9 cIAPI binding in vitro 150 ng mL His tagged PCSK9 wild type was used PCSK9 cIAP1 Binding Assay DMSO conc 6 Fig 4 Effect of NaCl on PCSK9 cIAPI binding jm v
9. ial containing 100 uL of 150X HRP conjugated anti His tag monoclonal antibody For long term storage 150X HiRP conjugated anti His tag monoclonal antibody solution can be stored in small aliquots e g 20 uL 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 Conjugate Dilution Buffer One bottle containing 15 mL of 1X buffet used for dilution of HRP conjugated anti His tag monoclonal antibody 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 mL of WN H2SO Ready to use Cat CY 8152 4 Version 150127 PCSK9 cIAP1 in vitro Binding Assay Kit CircuLex Users Maal For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor 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 Qy e Plate reader capable of measuring absor
10. ig 9 Effect of EGTA on PCSK9 cIAP1 binding in vitro 150 ng mL His tagged PCSK9 wild type AN was used PCSK9 cIAP1 Binding Assay EGTA conc 0 0 2 0 4 0 6 0 8 1 EGTA conc mM Fig 10 Effect of NP 40 on PCSK9 cIAP1 binding in vitro 1 g mL His tagged PCSK9 wild type was used PCSK9 cIAP1 Binding Assay NP 40 conc 0 2 0 3 NP 40 conc Y 8152 16 Version 150127 PCSK9 cIAP1 in vitro Binding Assay Kit C ircuLex y User s Manual For Research Use Only Not for use in diagnostic procedures References 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 Benjannef S Wickham L Erlich D et al 2003 Nat Genet 34 154 156 W 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 1264 41272 6 Berge KE Ose L Leren TP 2006 Arterioscler Thromb Vasc Biol 26 109471100 7 Maxwell KN Breslow JL 2004 Proc Natl Acad Sci USA 101 7100 7405 oo Rashid S Curtis DE Garuti R Anderson NN Bashmakov Y Ho YK Hammer RE Moon YA
11. ith recombinant human cIAP1 which contains binding site for PCSK9 HRP conjugated anti His tag monoclonal antibody specifically reacts with recombinant His tagged PCSK9 that has been trapped With recombinant human cIAP1 immobilized on the microplate surface The CircuLex PCSK9 cIAP1 in vitro Binding Assay Kit may be used to characterize PCSK9 cIAP1 interaction ag wells as to screening inhibitors of PCSK9 cIAP1 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 human cIAP1 immobilized on the wells After wash the plate the amount of bound recombinant His tagged PCSK9 is measured by binding it with HRP conjugated anti His tag monoclonal antibody 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 stepping reagent The color is quantitated by spectrophotometry and reflects the relative amount of fec mbinant His tagged PCSK that binds to recombinant cIAP1 on the microplate For screening inhibitors of PCSK9 cIAP1 interaction in vitro test compounds or inhibitor candidates including monoclonal antibodies are added to appropriate amount of His tagged PCSK9 in the wells that have b en coated with recombinant cIAPI in a similar fashion as described above followed by evaluation of nhibitory effect on PCSK9 cIAPI interaction by
12. itro 150 ng mL His tagged PCSK9 wild type was used PCSK9 cIAP1 Binding Assay NaCl conc 150 NaCl conc mM Cat CY 8152 13 Versions 150127 PCSK9 cIAP1 in vitro Binding Assay Kit CircuLex Cser Maal For Research Use Only Not for use in diagnostic procedures Fig 5 Effect of calcium ion on PCSK9 cIAP1 binding in vitro 50 ng mL His tagged PCSK9 wild 4 type was used e PCSK9 cIAP1 Binding Assay 0 5 ec T 0 0 1 1 1 1 J CaCl conc mM Fig 6 Effect of magnesium ion on PCSK9 cIAP1 binding in pitro 150 ng mL His tagged PCSK9 wild type was used PCSK9 cIAP1 Binding Assay MgCL conc 20 MgCl conc mM Y 8152 14 Version 150127 PCSK9 cIAP1 in vitro Binding Assay Kit C ircuLex y User s Manual For Research Use Only Not for use in diagnostic procedures Fig 7 Effect of BSA on PCSK9 cIAPI binding in vitro 150 ng mL His tagged PCSK9 wild type was AN used e PCSK9 cIAP1 Binding Assay BSA conc 0 2 0 3 BSA conc Fig 8 Effect of EDTA on PCSK9 cIAP1 binding ini O0 ng mL His tagged PCSK9 wild type was used PCSK9 cIAP1 Binding Assay EDTA conc EDTA conc mM Y 8152 15 Version 150127 PCSK9 cIAP1 in vitro Binding Assay Kit C ircuLex i User s Manual For Research Use Only Not for use in diagnostic procedures F
13. lly diluted His tagged PCSK9 wild type Solutions 150 2 34 ng mL afd Reaction Buffer only W Pipette 100 uL of the His tagged PCSK9 wild type Solutions 150 2 34 ng mL dnd Reaction Buffer only in duplicates into the wells Cover with plate sealer or lid and incubate at room 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 HRP conjugated anti His tag monoclonal antibody into each well cover with plate sealer or lid and incubate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker 7 Wash 4 times as same as in step 4 oo 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 9 Incubate the wells at room temperature ca 25 ifor 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 reaction temperature 1s below than 20 C 10 Add 100 uL of Stop Solution to each well in the Same order as the previous
14. ly added Substrate Reagent 11 Measure absorbance in each well usingga 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 liqui 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 cl an paper towels Note 2 Reliable signals ate obtained when either O D values do not exceed 0 25 units for the blank Reaction Buffer only or 3 0 units for t he 150 ng of His4agged PCSK9 wild type Solution Cat CY 8152 8 Version 150127 PCSK9 cIAP1 in vitro Binding Assay Kit C ircuLex y 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 cIAP1 interaction In order to estimate the inhibitory effect on PCSK9 cIAP1 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 4 st compounds cause an inhibitory effect on PCSK9 cIAPI interaction the level of A450 is weakened as compared with Vehicle Control
15. measuring the amount of His tagged PCSK9 On the wells Cat CY 8152 2 Versions 150127 PCSK9 cIAP1 in vitro Binding Assay Kit C ircuLex y User s Manual For Research Use Only Not for use in diagnostic procedures Schematic Representation of the CircuLex PCSK9 cIAP1 in vitro Binding Assay 4 Kit Principle amp e x mJ wash wash T cIAP1 Y HRP conjugated anti His tag mAb 3 TMB His tagged PCSK9 Summary of Procedure Add 100 uL of His tagged PCSK9 to the wells Incubate for 2 hrs at room temp ells f HRP conjugated anti His tag monoclonal Antibody Incubate for 1 hr at room temp Wash M wells e 100 uL M Substrate Reagent Add 100 uL of Solution i Measure absorbance at 450 nm Y 8152 3 Version 150127 PCSK9 cIAP1 in vitro Binding Assay Kit C ircuLex y 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 coated with recombinant human cIAP1 Recombinant His tagged PCSK9 wild type Two vials containing 0 9 ug of lyophilized recombinant His tagged PCSK9 wild type 150X HRP conjugated anti His tag monoclonal antibody One v
16. r mp PCSK9 cIAP1 in vitro Binding Assay Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Non Radioisotopic Assay Kit for Measuring PCSK9 cIAP1 interaction in vitro CircuLex PCSK9 cIAPI in vitro Binding Assay Kit Cat CY 8152 Intended Use sse 1 SLOT UH edes inu dioi RED REN l Introduction cccsecsccccececeesesssseceeeeeceesenes 2 Principle of the Assay 2 3 Summary of Procedure 3 Materials Provided ssssss 4 Materials Required but not Provided 5 Precautions and Recommendations 6 Detailed Protocol eeesesss 7 9 Evaluation of Results s 10 Troubleshooting seossstnse pon ORT enia 11 Reagent Stability aai s irrito tinta 11 Assay Characteristics o 12 16 References ssseseseeeeeeeeeenennnennnne 17 Related Product esses 17 Intended Use The CycLex Research Product CircuLex PCSK9 eIAP1 in vitro Binding Assay Kit is primarily designed to screening inhibitors of PCSK9 cIAPI 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 cIAPI interaction in vitro The recombinant human cIAP1 used in this assay kit was designed for keeping the correct conformation to bind PCSK9 even if immobilized on the mic
17. roplate surface This technique allows us measuring efficiently the interaction of recombinant PCSK9 to recombinant cIAP1 in a solid phase assay system such as a conventional ELISA system Applications for this kit include 1 Screening inhibitors ofPCSK9 eIAP1 interaction in vitro 2 Characterization of inhibitof candidates of PCSK9 cIAP1 interaction in vitro 3 Screening monoclonal antibody that inhibits PCSK9 cIAPI interaction in vitro 4 Characterization of PCSK9 cIAP1 interaction in vitro This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage e Upon re eipt store all components at 4 C Dom ttexpose reagents to excessive light Cat CY 8152 1 Versions 150127 PCSK9 cIAP1 in vitro Binding Assay Kit C ircuLex y 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 hypercholesterolefnia ADH These studies suggested that gain of function mutations increase plasma levels of LIDL c 2 6 whereas nonsense or missense loss of function mutations which interfere withefolding or secretion
18. ter plate readers perform automatic calculations of afialyte feoncentration The calibration curve is constructed by plotting the absorbance Y of calibrators 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 e logit of absorbance Y is plotted versus log of the known concentration X of calibrators Note The CycLex Research Product CircuLex PCSK9 cIA PT in vitro Binding Assay Kit has been shown to measure PCSK9 cIAPI interaction in vitro The assay may be used to screening and characterize inhibitors of PCSK9 cIAPI interactioibin vitro Please consider whether your experimental condition is appropriate by drawing a direct line with the results of Assay Characteristics in page 12 16 Cat CY 8152 10 Version 150127 PCSK9 cIAP1 in vitro Binding Assay Kit C ircuLex y User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting 1 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 significan different from those specified may give erroneous results 2 Poor duplicates accompanied by elevated values for wells containing no sample indicate insufficient washing If all instructions in the Detailed Protocol were followed acc results indi
19. ug mL which is referred as a Master solution of Recombinant His tagged PCSK9 wild type 3 Prepare His tagged PCSK9 wild type Solutions by 2 fold serially diluting the Master solution 1 5 ug mL to an appropriate concentration usually4 50 0778 ng mL only for drawing dose response curve with Reaction Buffer See the section Standard Assay for Drawing Dose Response Curve in page 8 at the time of assay 4 Prepare 1X HRP conjugated anti His tag monoclonal antibody by diluting the 150X HRP conjugated anti His tag monoclonal antibody solution 150 fold with Conjugate Dilution Buffer at the time of assay For an inhibitor screening assay the Master solution 1 5 ug mL should be diluted with Reaction Buffer to appropriate concefitration which shows OD value does not exceed plateau range in dose response curve Prepare appropriate volume for youf assay Discard any unused Recombinant His tagged PCSK9 Solutions and 1X HRP conjugated anti His tag monoclonal antibody after diluted Cat CY 8152 T Version 150127 PCSK9 cIAP1 in vitro Binding Assay Kit C ircuLex y 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 N Prepare 2 fold seria
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