CircuLex AGE-RAGE in vitro Binding Assay Kit
Contents
1. e AGE2 BSA coated plate my o BSA coated plate of control 0 0 0 2 0 4 0 6 0 8 1 0 1 2 1 4 1 6 1 8 2 0 BSA concentration o gL eA Roa CY 8151 16 Version 150817 amp lt L Reva CY 8151 17 Version 150817 CircuLex Users Manual References Neeper M Schmidt AM Brett J et al Cloning and expression of a cell surface receptor foxg anced j 9 10 11 12 A S AGE RAGE in vitro Binding Assay Kit For Research Use Only Not for use in diagnostic procedures 4 glycosylation end products of proteins J Biol Chem 1992 267 14998 15004 RY Brett J Schmidt AM Yan SD et al Survey of the distribution of a newly characteriggg receptor for advanced glycation end products in tissues Am J Pathol 1993 143 1699 1712 N Schmidt AM Vianna M Gerlach M et al Isolation and characterization of tw binding proteins for advanced glycosylation end products from bovine lung which are presentygn the endothelial cell surface J Biol Chem 1992 267 14987 14997 Hori O Brett J Slattery T Cao R et al The receptor for advanced gly in end products RAGE is a cellular binding site for amphoterin Mediation of neurite outgrow d coexpression of rage and amphoterin in the developing nervous system J Biol Chem 1995 27Q3 5752 25761 endproduct interaction elicits neuronal expression of m hage colony stimulating factor a proinflammatory pathway i2. Reagent Stability All of the reagents included in the CycLex Research Pog CircuLex AGE RAGE in vitro Binding Assay Kit have been tested for stability Reagen ould not be used beyond the stated expiration date Reva CY 8151 12 Version 150817 o c A CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures y AGE RAGE in vitro Binding Assay Kit neers Assay Characteristics A Fig 1 Typical dose dependent curve of Recombinant His tagged sRAGE D AGE RAGE in vitro Binding Assay AGE2 BSA coated plate S BSA coated plate 2 0 F A450 1 0 0 0 0 20 40 60 80 esRAGE 8His ng ml Fig 2 Inhibition of AGE RAGE Binding in gto with Compound DP Inhibition with Control Compound Compound DP of control 0 1 10 100 Compound DP concentration uM A Reon CY 8151 13 Version 150817 S C AGE RAGE in vitro Binding Assay Kit CircuLex Users Manual Fig 3 Effect of pH on AGE RAGE Binding in vitro 80 ng mL His tagged sRAGE was used For Research Use Only Not for use in diagnostic procedures Effect of pH on AGE RAGE in vitro Binding Assay 120 100 L AGE2 BSA coated plate BSA coated plate 80 40 20 5 0 5 5 6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 pH v Fig 4 Effect of DMSO on AGE RAGE Binding ijro esr 4 J Effec
3. A Q O Q Materials Provided 3 All samples should be assayed in duplicate The following components are supplied and ar icient for the one 96 well microtiter plate kit RY AGE2 BSA coated Microplate One microplate supplied ready to use with 96 wel 9y 12 strips of 8 well in a foil zip lock bag with a desiccant pack Wells are coated amp AGE2 BSA glyceraldehyde modified BSA Y BSA coated Microplate One microplate supplied ready to use with 96 wells 2 strips of 8 well in a foil zip lock bag with a desiccant pack for non specific binding control Wells Qy coated with BSA Recombinant His tagged sRAGE One vial containing 1 6 ug of yopa recombinant His tagged human sRAGE Reaction Buffer One bottle containing 50 mL of 1X buffer used Por reconstitution and dilution of Recombinant His tagged sRAGE and binding reaction Ready to 20X Inhibitor Control Compound 250 uM compound DP which was discovered in commercial chemical library by CycLex using this CircuLex AGE RY E in vitro Binding Assay Kit This compound DP may be irritating to the skin eyes and uppePrespiratory tract and hazardous in case of ingestion This is also suspected to be carcinogenic PleaSeJhandle carefully according to Guidelines on Handling Chemical Carcinogens and Mutagens Y HRP conjugated anti His tag monoclonal antibody One bottle containing 30 mL of HRP horseradish peroxidase conjugated anti His tag bnoclonal antibody Ready to use 10X Wash B
4. CircuLex AGE RAGE in vitro Binding Assay Kit is pro 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 conditions may vary individua ter should optimize the amount of Recombinant His tagged sRAGE for inhibitor screening frongdose response curve Disposable pipette tips and reagent troughs should be used for all van to avoid cross contamination of reagents or samples Y I Preparation of Working Solutions y 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10 ash Buffer provided to 900 mL of deionized water Mix well Store at 4 C for two weeks or KN for long term storage action Buffer After dissolved L to micro centrifuge tubes and concentration of the reconstituted s referred as a Master solution of 2 Reconstitute the Recombinant His tagged sRAGE with 0 8 mL o by gently mixing immediately dispense in small aliquots e g store at below 70 C to avoid repeated freezing and thawing F recombinant His tagged sRAGE should be 2 ug mL whi Recombinant His tagged sRAGE 3 Prepare His tagged sRAGE Solutions by 2 fold seriall uting the Master solution 2 ug mL to an appropriate concentration usually 100 1 56 ng mL nly for drawing dose response curve with Reaction Buffer at the time of assay See II S d Assay for Drawing Dose Response Curve below Note 1 For an inhibitor s
5. and 20X Test Compound or Vehicle for Inhibitor or 20X Inhibitor Control compound to each well of the AGE2 BSA coated Microplate and BSA coated Microplate 2 Initiate reaction by adding 10 uL of 10x His tagged sRAGE solution to each well and mixing thoroughly Cover with plate me lid and incubate at room temperature for 1 hour shaking at ca 300 rpm on an orbital microplat er 3 Follow the steps 4 10 of Mean Assay for Drawing Dose Response Curve above Note 1 The optimal rimental conditions must be determined by the individual user Especially appropriate entration of 10X His tagged sRAGE Solution must be determined by drawin response curve which shows OD value does not exceed plateau range in the dose x a Ya curve Note 2 To obtain an accurate inhibition percentage of test compounds draw and calculate form a sg one curve with appropriate serial dilutions of 10X His tagged sRAGE Solution in p el with the inhibition assay of the test compounds Note 3 dmhibition percentage of Inhibitor Control Compound 12 5 uM compound DP provided S this kit should be more than 40 See Fig 2 in the section Assay Characteristics Q below NO THE ABOVE PROCEDURES ARE INTENDED ONLY AS A GUIDELINE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY DEPENDING ON THE PARAMETERS c BEING INVESTIGATED NO WARRANTY OR GUARANTEE OF PERFORMANCE S USING THESE PROCEDURES IS MADE OR IMPLIED LA at CY 8151 10 Version 15
6. of calibrators versus log of the known concentration X of calibrators using the four gayameter function Alternatively the logit log function can be used to linearize the calibratio Mwe i e logit of absorbance Y is plotted versus log of the known concentration X of cava Note The CycLex Research Product Crap SAGE RAGE in vitro Binding Assay Kit has been shown to measure AGE RAGE int ion in vitro The assay may be used to screen and Ae eni PON s sane interaction in vitro Please _consider__whether your a i ith Reva CY 8151 11 Version 150817 amp c A CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures 4 Troubleshooting 3 All samples should be run in duplicate when a standard assay is being performed using t otocol described in the Detailed Protocol Incubation times or temperatures significantly di nt from those specified may give erroneous results S AGE RAGE in vitro Binding Assay Kit j 2 Poor duplicates accompanied by elevated values for wells containing no sample i te insufficient washing If all instructions in the Detailed Protocol were followed accurately results indicate a need for washer maintenance Q 3 Overall low signal may indicate that desiccation of the plate has occurred ben the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add S te Reagent immediately after wash Y
7. product without prior written approval from CycLex Co Ltd To inquire about licensing for suepi mmercial use please contact us via email A Reon CY 8151 18 Version 150817 o c
8. room temperature ca 25 C for 1 hour shaking at c 0 rpm on an orbital microplate shaker Y 6 Wash 4 times as same as in step 4 sunlight Covering the wells with e g aluminum foil is recommended Return Su e Reagent to 7 Add 100 uL of Substrate Reagent into each well Avoid exposing the microtiter plate to direct 4 C immediately after the necessary volume is removed i Y Incubate the wells at room temperature ca 25 C for 5 15 minutes shaki ca 300 rpm on an orbital microplate shaker Appropriate incubation time may vary an ubation time can be extended up to 30 minutes if the reaction temperature is below than 20AN co 9 Add 100 uL of Stop Solution to each well in the same order as previously added Substrate Reagent yy 10 Measure absorbance in each well using a spectrophotometri te reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm also be used Read the plate at 450 nm if only a single wavelength can be used Wells muy read within 30 minutes of adding the Stop Solution A Note 1 Complete removal of liquid at each step i ential to good performance After the last wash remove any remaining Wash Butte by aspirating or decanting Invert the plate and blot it against clean paper towels lt lt Note 2 Reliable signals are obtained when xo O D values do not exceed 0 25 units for the blank Reaction Buffer only or 3 0 unit or the 100 ng mL of Recombinant His tagged sR
9. 0817 o c AGE RAGE in vitro Binding Assay Kit CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures A Q O Q SS Evaluation of Results 3 To obtain an accurate inhibition percentage of test compounds it s required to draw an culate form a dose response curve with serial dilutions of 10X His tagged sRAGE Solution in par with the inhibition assay of the test compounds 1 Average the duplicate readings for each Inhibitor Control Test Compound ehicle Control and serial dilutions of 10X His tagged sRAGE Solution in AGE2 BSA coated coated Microplate Subtract the average readings in BSA coated Microplate fro coated Microplate The absorbance value for Vehicle Control in AGE248SA coated Microplate should be greater than 1 0 with a non specific signal in BSA coated meee which should be less than 0 25 Y Inhibition percentage of test compounds should be calculated from thea yse response curve The dose response curve of this assay fits to a sigmoidal four paraffeter logistic equation The results of unknown samples can be calculated with any computer pro having a four parameter logistic function It is important to make an appropriate mathe 1 adjustment to accommodate for inhibition percentage lt Most microtiter plate readers perform automatic calculatfgps of analyte concentration The calibration curve is constructed by plotting the absorbance J
10. A P ihi AGE RAGE in vitro Binding Assay Kit C 1rcu Lex User s Manual A For Research Use Only Not for use in diagnostic procedures Q gt Non Radioisotopic Kit for Measuring AGE RAGE Interaction in vitro l CircuLex AGE RAGE in vitro Binding Assay Kit amp Cat CY 8151 2 Intended Use ccccccccccccccececeeeseeeeseees 1 oO 1a 1 A eee ae emer aera nae peeve Weert 1 aS Introduction 00 0 0 eecccccceeseessssesssssseeseees 2 rw Principle of the Assay 2 3 y Summary of Procedure 4 2 Materials Provided cccceeeeeeeeeeeeeeees 5 oO Materials Required but not Provided 6 N Precautions and Recommendations 7 lt Detailed Protocol ccccccccccceessssseeeeeeeeeees 8 10 eA Evaluation of Results ccc ceeeeeeeeee ee 11 RZ Troubleshooting gece ene eer 12 Reagent Stability cecisantencdonsavensacoanssseeanceoaies 12 Assay Characteristics s 1 ReferetC Ssnccncinniranininan nnana 19 Related Product nnnnennnneneng I s ntended Use The CycLex Research Product ciret Pex AGE RAGE in vitro Binding Assay Kit is primarily designed to screening inhibitors of AQF2 glyceraldehyde modified AGE sRAGE interaction soluble RAGE in vitro In addition this 6h can be used for the rapid and sensitive evaluation of inhibitor candidates as well as characterizd jn of AGE2 sRAGE interaction in vitro The recombinant sRAGE used in this assay kit was specit designed by CycLex for keeping th
11. AGE Roa CY 8151 9 Version 150817 o AO A AGE RAGE in vitro Binding Assay Kit User s Manual AS For Research Use Only Not for use in diagnostic procedures Q CircuLex gt II Standard Assay for Inhibitor Screening l Special considerations when screening inhibitors of AGE RAGE interaction RA In order to estimate the inhibitory effect on AGE RAGE interaction in the test compounds efectly it is necessary to conduct the control experiment of Vehicle Control for every experiment Inhibitor Control at least once for the first experiment in addition to Test Compound as igglicated in the following table When test compounds cause an inhibitory effect on AGE RAGE inter n the level of A450 is weakened as compared with Vehicle Control PN For inhibitor screening Mag Inhibitor ae Vehicle Assay Reagents Control ompound Control Reaction Buffer 85 uL 5 uL 85 uL 20X Inhibitor Control Compound 5 uL 20X Test Compound s 5 uL Vehicle for 20X Test Compound s 5 uL 10X His tagged sRAGE Solution 10g 10 pL 10 pL 20X Inhibitor Control Compound See the section Matgjjals Provided above 10x concentrated solution of the His tagged s Solution which shows OD value does not exceed plateau range in dose response curva suay 500 750 ng mL see below See I Preparation of Working Solutions above 1 Following the above table add the Reactidhwbuffer
12. AGE RAGE in vitro Binding Assay Kit Circu Lex User s Manual A For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations 3 e Allow all the components to come to room temperature before use eS ws e All microplate strips that are not immediately required should be returned to the zip lock Xa which must be carefully resealed to avoid moisture absorption Q e Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit Meg e Rinse all detergent residue from glassware Rd e Use deionized water of the highest quality gt e Do not mix reagents from different kits 2 The buffers and reagents in this kit may contain preservatives V chemicals Care should be taken to avoid direct contact with these reagents lt Q e Do not smoke eat or drink when performing the yay or in areas where samples or reagents are handled aS e Dispose of tetra methylbenzidine TMB contas solutions in compliance with local regulations Do not mouth pipette or ingest any of the reagents e CAUTION Sulfuric Acid is a stron g Wear disposable gloves and eye protection when handling Stop Solution A A Roa CY 8151 T Version 150817 e ee AGE RAGE in vitro Binding Assay Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Detailed Protocol 3 The CycLex Research Product
13. CycLex Research Products Ci ex AGE RAGE in vitro Binding Assay Kit is a semi quantitative solid phase binding ads between recombinant His tagged soluble RAGE SRAGE and glyceraldehyde modified BSA 2 BSA Plates are pre coated with AGE2 BSA which is able to bind to recombinant His tageed QRAGE Horse radish peroxidase HRP conjugated anti His tag monoclonal antibody specifica dey with recombinant His tagged sRAGE that is trapped with AGE2 BSA immobilized on t croplate well surface The CircuLex AGE RAGE in vitro Binding Assay Kit may be used t aracterize AGE RAGE interaction as well as to screen inhibitors of AGE RAGE interaction NiO To perform the test combinant His tagged sRAGE is diluted in Reaction Buffer pipetted into the wells and allowed t to AGE2 BSA immobilized on the wells After wash the wells the amount of bound recombin s tagged sRAGE is measured by binding it with HRP conjugated anti His tag monoclonal antibody which then catalyzes the conversion of the chromogenic substrate tetra B from a colorless solution to a blue solution or yellow after the addition of The color is quantitated by spectrophotometry and reflects the relative amount of tagged sRAGE that binds to AGE2 BSA on the wells Reva CY 8151 2 Version 150817 o c CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures S AGE RAGE in vitro Binding Assay Kit For screening inhibitors of AGE RAGE interact
14. creening assay 4P Master solution 2 g mL should be diluted with Reaction Buffer to appropriate concentration which shows OD value does not exceed plateau range in dose response ve Note 2 Prepare appropriate volute for your assay Discard any unused His tagged sRAGE Solutions after diluted amp II Standard Assay for Dra f g Dose Response Curve 1 Remove the appropriate amp amp mber of microtiter wells of AGE2 BSA coated Microplate from the foil pouch and place then ijto the well holder Return any unused wells to the foil pouch refold seal with tape and store C 2 Prepare 2 fold NY diluted His tagged sRAGE Solutions 100 1 56 ng mL and Reaction Buffer only rd 3 Pipette 10 of the His tagged sRAGE Solutions 100 1 56 ng mL and Reaction Buffer only in duplica into the wells Cover with plate sealer or lid and incubate at room temperature cae Bior 1 hour shaking at ca 300 rpm on an orbital microplate shaker 4 4 times by filling each well with Wash Buffer 350 uL using a squirt bottle multi channel ette manifold dispenser or microplate washer Remove residual Wash Buffer by gentle tapping Cor aspiration Add 100 uL of HRP conjugated anti His tag monoclonal antibody into each well cover with at CY 8151 8 Version 150817 A CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures S AGE RAGE in vitro Binding Assay Kit lt plate sealer or lid and incubate at
15. e correct conformation to bind efficiently to AGE2 BSA not to BSA which are immobilized on the microplate surface This technique allows us measwging the interaction of recombinant sRAGE to AGE2 BSA in a solid phase assay system such as a ntional ELISA system Applications for eet 1 Screening inhibit rs of AGE sRAGE interaction in vitro 2 Characteriza ZA of inhibitor candidates of AGE2 sRAGE interaction in vitro 3 Screening g ehoclonal antibodies that inhibit AGE2 sRAGE interaction in vitro 4 Characte hedtion of AGE2 sRAGE interaction in vitro aA store all other components at 4 C DG t expose reagents to excessive light Roa CY 8151 1 Version 150817 o c AGE RAGE in vitro Binding Assay Kit L ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 A Q O Q Introduction 3 Advanced glycation end products AGEs are important biochemical compounds found gh abetes and may be associated with inflammatory processes In the vessel wall AGEs are bound te specific receptors to modulate many cellular properties by activating several signaling pathways ae of these receptors is called Receptor for Advanced Glycation End product RAGE RAGE is a multi ligand member of the immunoglobulin superfamily of cell surfac lecules that is expressed in a variety of cell lines including endothelial cells smooth muscle s mononuclear phagocytes pericytes neurons cardiac myocyte
16. ion in vitro test compounds or the inhMjtor candidates including a monoclonal antibody are added to appropriate amount of His tagged RAGE i in the wells pre coated with AGE2 BSA in a similar fashion as described above followed by e tion of inhibitory effect on AGE RAGE interaction by measuring the amount of His tagged s 2 on the wells S Schematic Representation of the CircuLex AGE RAGE in iada Assay Kit Principle s Schematic Representation of AGE SAGE in vitro Binding Assay Kit Prigfciple g at amp g wash wash Q y 9 AGE2 BSA O HRP conjugated anti His tag mAb amp ape A z Py O A a at CY 8151 3 Version 150817 S C ww P ihi AGE RAGE in vitro Binding Assay Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Assay Procedure 3 Add 100 uL of recombinant His tagged sRAGE to the wells we 4 Incubate for 1 hour at room temp Y Wash the wells 74 4 Q Add 100 uL of HRP conjugated Anti His tag monoclonalggribody 4 Incubate for 1 hour at room y Wash the wells ac gt wW Y gt Add 100 uL of Substrate Reagent wy Add 100 uL of Stop Solution N t A Measure absorbance at 450 nm KG 2 Roa CY 8151 4 Version 150817 amp lt L AGE RAGE in vitro Binding Assay Kit CircuLex Users Manual For Research Use Only Not for use in diagnostic procedures 4
17. n Alzheimer disease Proc Natl Aga Sci US A 1997 94 5296 5301 Yan SD Zhu H Fu J Yan SF Roher A et al Amyloidbeta Sect for advanced glycation Yan SD Zhu H Zhu A Golabek A Du H Roher A Con J Soto C Schmidt AM Stern D et al Receptor dependent cell stress and amyloid accumulatigfi in systemic amyloidosis Nat Med 2000 6 643 651 Q Chavakis T Bierhaus A Al Fakhri N et ihe pattern recognition receptor RAGE is a counterreceptor for leukocyte integrins a ge pathway for inflammatory cell recruitment J Exp Med 2003 198 1507 1515 4 Sasaki N Takeuchi M Chowei H Kiku et al Advanced glycation end products AGE and their receptor RAGE in the brain of pati with Creutzfeldt Jakob disease with prion plaques Neurosci Lett 2002 326 117 120 Chapman MR Robinson LS Pyakner JS et al Role of Escherichia coli curli operons in directing amyloid fiber formation SOO 2002 295 851 855 Bierhaus A Humper R Morcos M et al Understanding RAGE the receptor for advanced glycation end productsQ ol Med 2005 83 876 886 Hofmann MA Fu C Qu W et al RAGE mediates a novel proinflammatory axis a central cell surface rece for 100 calgranulin polypeptides Cell 1999 97 889 901 Stern DM YOR Yan SF and Schmidt AM Receptor for advanced glycation endproducts RAGE and the complications of diabetes Ageing Res Rev 2002 1 1 15 Q amp amp o c P ihi AGE RAGE in vitro Bi
18. nding Assay Kit C 1rcu Lex User s Manual AS For Research Use Only Not for use in diagnostic procedures Related Products 3 CircuLex S100A12 EN RAGE ELISA Kit Cat CY 8058 we CircuLex Human sRAGE ELISA Kit Cat CY 8083 N CircuLex AGE RAGE in vitro Binding Assay Kit Cat CY 8151 KY RAGE HEK293 Cell Line Cat CY C8250 g Anti Human RAGE Clone YK 2B4 Cat CY M1038 2 CML BSA N Carboxymehtyl lysine BSA Cat CY R2052 oO CML OVA N Carboxymehtyl lysine OVA Cat CY R2053 ne CEL BSA N Carboxyehtyl lysine BSA Cat CY R2054 a CEL OVA N Carboxyehtyl lysine OVA Cat CY R2055 Y Glucose AGE BSA Cat CY R2056 wy Glucose AGE OVA Cat CY R2057 amp Glyceraldehyde AGE BSA Cat CY R2058 Yon Glyceraldehyde AGE OVA Cat CY R2059 Glycolaldehyde AGE BSA Cat CY R2060 S Glycolaldehyde AGE OVA Cat CY R2061 amp Methylglyoxal AGE BSA Cat CY R2062 Methylglyoxal AGE OVA Cat CY R2063 X Glyoxal AGE BSA Cat CY R2064 amp Glyoxal AGE OVA Cat CY R2065 ny CML HSA N Carboxymehtyl lysine HSA Cat CY R2066 CEL HSA N CarboxyehtyD lysine HAS bi CY R2067 PRODUCED BY Ina Nagano 396 00 Japan Fax 81 265 76 518 E mail info c ex co j CycLex Co Ltd AY 1063 103 no eS Cycle ifr ex products are supplied for research use only CycLex CircuLex products and comp s thereof may not be resold modified for resale or used to manufacture commercial
19. s mesangial cells and hepat s 1 2 RAGE interacts with different structures to transmit a signal into the cell and recoggizes three dimensional structures rather than specific amino acid sequences Therefore RAGE seems Oyu the requirements of a pattern recognition receptor As a member of the immunoglobulin s mily it interacts with a diverse class of ligands including AGEs 1 3 HMGB1 also knownas Amphoterin 4 amyloid B peptide 5 amyloid A 6 leukocyte adhesion receptors 7 prions 8 cherichia coli curli operons 9 B sheet fibrils 10 and several members of the S100 yotein superfamily including 100 calgranulins 11 Thus RAGE may have potential involvemes n several pathological processes including inflammation diabetes Alzheimer s disease AD syst ftic amyloidosis and tumor growth 12 AGE RAGE interaction enhances the expression of a Seige cytokines growth factors and adhesion molecules and increases the classical acute phase wy eins Potential approaches to prevention and treatment of diabetes and its complications includ ibition of AGE formation breakage of preformed AGE proteins crosslink blockade of Rae ae interactions with RAGE competitors antibody antagonists and RAGE specific metabolic phibition Inhibition of AGE RAGE complex formation is able to suppress the levels of ee cytokines and growth factors and may be considered as a target for overcoming diabetic comph ations Principle of the Assay The
20. t of DMSO on AGE RAGE in vitro Binding Assay 120 100 a a 80 es 60 AGE2 BSA coated plate 40 s BSA coated plate 20 P64 6 _9 i S o D 0 0 0 1 0 2 0 3 0 4 0 5 0 DMSO concentration oe Reva CY 8151 14 Version 150817 o c A P ihi AGE RAGE in vitro Binding Assay Kit CircuLex Users Manwal lt For Research Use Only Not for use in diagnostic procedures Fig 5 Effect of Calcium Ion on AGE RAGE Binding in vitro 40 ng mL His tagged sRAGE was Ey Effect of Ca2 on AGE RAGE in vitro Binding Assay 120 100 80 60 AGE2 BSA coated plate BSA coated plate 40 20 0 0 100 200 300 400 500 600 700 800 900 1000 CaCl2 concentration mM Ww Fig 6 Effect of NaCl on AGE RAGE Binding in log 40 ng mL His tagged sRAGE was used fey Effect of NaCl on AGE RAGE in vitro Binding Assay 120 100 80 60 e AGE2 BSA coated plate o BSA coated plate 0 100 200 300 400 500 600 700 800 900 1000 NaCl concentration mM Roa CY 8151 15 Version 150817 o c ihi AGE RAGE in vitro Binding Assay Kit C 1rcu Lex User s Manual A For Research Use Only Not for use in diagnostic procedures Q S RN Fig 7 Effect of BSA on AGE RAGE Binding in vitro 40 ng mL His tagged sRAGE was used Effect of BSA on AGE RAGE in vitro Binding Assay 120
21. uffer One bottle containing om of 10X buffer containing Tween 20 Substrate Reagent One bottle contain 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containipy 20 mL of 1 N H2804 Ready to use A lt S oO Q Qe amp A Reva CY 8151 5 Version 150817 o c A CircuLex Users Manual S AGE RAGE in vitro Binding Assay Kit For Research Use Only Not for use in diagnostic procedures 4 Materials Required but not Provided 3 e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips RS N Precision repeating pipettor S e Orbital microplate shaker g Wash bottle or multichannel dispenser for plate washing 2 e Microcentrifuge and tubes for sample preparation e Vortex mixer y D O e Microplate washer optional Manual washing is possible but not pre rable e Plate reader capable of measuring absorbance in 96 well plate dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also ed The plate can also be read at a single wavelength of 450 nm which will give a somewhat hggh r reading Optional Software package facilitating data analysis e 500 or 1000 mL graduated cylinder amp D O amp e Reagent reservoirs Deionized water of the highest quality e Disposable paper towels Roa CY 8151 6 Version 150817 o c RS P ihi
Download Pdf Manuals
Related Search