CY-8053 Rat NGAL/Lipocalin
Contents
1. Note Do not use a Repeating pipette Change tips for every dilution Wet tip with Dilution Buffer before dispensingsUnused portions of Master Standard should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Cat CY 8053 8 Version 140318 Tii Rat NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation e Serum and plasma samples usually require a 500 fold or 1 000 fold dilution with Dilution Buffer e Urine samples usually require a 100 fold to 5 000 fold dilution with Dilution Buffer Note 1 Please predict the concentration before carrying out assay If values for these are not within the range of the standard curve users must determine the optimal sample dilutions for their particular experiments Note 2 Dilutions lower than 10 fold should not be used Assay Procedure p Prepare the assay protocol assigning the appropriate wells for setting up_Standard Solutions diluted patient specimens and any internal laboratory controls in duplicate Removewthe appropriate number of microtiter wells from the foil pouch and place them into the well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 Dilute samples with Dilution Buffer See Sample Preparation above w Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted samples in duplicate2. Average the duplicate readings for each standard control and sample and subtract the average zero standard optical density Plot the optical density for the standards versus the concentration of the standards and draw the best curve To determine the rat NGAL concentration of each sample first find the absorbance value on the y axis and extend a horizontal line to the standard curve At the point of intersection extend a vertical line to the x axi and read the corresponding rat NGAL concentration If the samples have been diluted the concentrationfread from the standard curve must be multiplied by the dilution factor ja The dose response curve of this assay fits best to a sigmoidal 4 parameter logistic equation The results of unknown samples can be jcalculated with any computer program having a 4 parameter logistic function It is importantto make an appropriate mathematical adjustment to accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of analyte concentration The calibration curve is constructed y 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 knowWn cone entration X of calibrators 3 Dilution factors need to be taken into consideration in calcula
3. T Rat NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Rat NGAL Lipocalin 2 Yy CircuLex Rat NGAL Lipocalin 2 ELISA N Cat CY 8053 Intended Use cccccccccccccceceeeeeseeeeseees 1 SOO A 1 TntroductiOn ccccceecccccccecceeeeccceeseeeesseesees 2 Qy Principle of the Assay 3 Materials Provided cccccccccccceccsesseeeeceeees 4 Materials Required but not Provided 5 Precautions and Recommendation 6 Sample Collection and Storage T Detailed Protocol cccecccccccccccceeceeeeeeeeeeeens 8 10 Calculations srera enero a 10 Measurement Ratige is cccsssvvacctansviesveeeness 11 Troubleshooting sseeeeeeeseseeeseeereserersrssee 11 Reagent Stability vcicsssicsscchsistvsavaressiscpeieaiende 11 Assay Characteristics c ciccsscesssssssesesessvees 12 Example of Test Results cee eeeeeeeeeees References isis sinc scisatedienctieeseed RONNA Related Products cccccceeesseesseeeeeeeeeees 16 Intended Use The CycLex Research Product CircuLex Rat NGAL Lipocalin 2 ELISA Kit is used for the quantitative measurement of rat NGAL calin 2 in serum plasma urine tissue culture medium and other biological media This assay kit is for research use o lya d not for use in diagnostic or therapeutic procedures Storage e Upon receipt store all co Don t expose reagents C CY 80
4. the reconstituted rat NGAL Standard must be stored at below 70 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack Cat CY 8053 11 Version 140318 Rat NGAL Lipocalin 2 ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of rat NGAL giving absorbance higher than mean absorbance of blank plus three standard deviations of the absorbance of blank A blank 3SD blank is better than 6 02 pg ml of sample Dilution Buffer is pipetted into blank wells 2 Precision Intra assay Precision Precision within an assay Three samples of known concentration were tested sixteen times on one plate fOjassess intra assay precision e Intra assay Within Run n 16 CV 2 4 2 3 4 4 Rat NGAL cones ng mill Cat CY 8053 12 Version 140318 Rat NGAL Lipocalin 2 ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Inter assay Precision Precision between assays Three samples of known concentration were tested in five separate assays to assess inter assay precision e Inter assay Run to Run n 5 CV 3 7 8 9 6 7 Rat NGAL conc ng ml Day Serum 1 Serum 2 Serum 3 1 663 3 376 0 110 9 2 606 1 345 1 97 2 3 612 5 316 5 106 9 4 635 5 395 7 115 7 5 644 4 337 0 10
5. 0 20 minutes shaking at ca 300 rpm onan orbital microplate shaker The incubation time may be extended up to 30 minutes if the reaction temperature is below than 20 C 14 Add 100 uL of Stop Solution to each well in the same order as the previously added Substrate Reagent 15 Measure absorbance in each well using a spectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the miefoplate 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 Invertthe plate and blot it against clean paper towels Note 2 Reliable standard curves are obtained when either O D values dogifot ex eed 0 2 units for the blank zero concentration or 2 5 units for the highest standard Concentration The plate should be monitored at 5 minute intervals for approximately 30minutes Note 3 If the microplate reader is not capable of reading absorbanc greater than the absorbance of the highest standard perform a second reading at 405 nm4A new standard curve constructed using the values measured at 405 nm is used to determine rat NGAL concentration of off scale samples The readings at 405 nm should not teplace the on scale readings at 450 nm Calculations
6. 3 CircuLex Human NGAL ELISA Kit Cat CY 8070 CircuLex Mouse FABP1 L FABP ELISA Kit Cat CY 8054 Coming soon CircuLex Mouse FABP3 H FABP ELISA Kit Cat CY 8055 Coming soon CircuLex Mouse FABP5 E FABP ELISA Kit Cat CY 8056 Coming soon CircuLex Mouse FABP4 A FABP ELISA Kit Cat CY 8077 CircuLex Rat FABP4 A FABP ELISA Kit Cat CY 8076 CircuLex Rat Adiponectin ELISA Kit Cat CY 8049 CircuLex Human Adiponectin ELISA Kit Cat CY 8050 CircuLex Mouse Adiponectin ELISA Kit Cat CY 8051 CircuLex Dog Adiponectin ELISA Kit Cat CY 8052 CircuLex S100A13 ELISA Kit Cat CY 8057 CircuLex S100A12 EN RAGE ELISA Kit Cat CY 8058 CircuLex S100A4 ELISA Kit Cat CY 8059 CircuLex S100P ELISA Kit Cat CY 8060 CircuLex S100A8 MRP8 ELISA Kit Cat CY 8061 CircuLex S100A9 MRP14 ELISA Kit Cat CY 8062 CircuLex S100A7 Psoriasin ELISA Kit Cat CY 8073 CircuLex CML Ne carboxymethyl Lysine ELISA Kit Cat CY 8066 CircuLex High Sesitivity CRP ELISA Kit Cat CY 8071 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyelex cogp URL http www cyclex e0 jp CycLex CircuLex 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 products without prior written approval from CycLex Co Ltd To inquire about licensing for such c
7. 3 0 MAX 663 3 395 7 115 7 MIN 606 1 316 5 97 2 MEAN 632 4 354 1 1067 S D 23 415 31 592 7 116 C V 3 70 8 92 6 67 3 Spiking Recover Serum samples were spiked with different amounts of rat NGALjand assayed The recovery of rat NGAL spiked to levels throughout the ra ge ofthe assay was evaluated Sample Average Recovery Range Cell culture media n 4 92 87 98 109 4 Linearity To assess the linearity of the assay samples containingyand or spiked with high concentrations of rat NGAL were serially diluted with the Dilution Buffer to produce samples with values within the dynamic range of the assay Linearity 1 0 gt Rat NGAL conc ng ml gt on 0 3 0 0 0 0 2 0 4 0 6 0 8 1 1 2 1 4 Dilution ratio 100 Cat CY 8053 13 Version 140318 Rat NGAL Lipocalin 2 ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Typical standard curve Rat NGAL Standard Curve 25 ne se A450 0 0 25 0 5 0 75 1 1 25 Rat NGAL conc ng ml References ji Kjeldsen L Johnsen AH Sengelov H Borregaard N 1993 Isolation and primary structure of NGAL a novel protein associated with human eutrophil gelatinase J Biol Chem 268 10425 32 2 Devireddy L R Teodoro J G Riehard EF A and Green M R 2001 Induction of apoptosis by a secreted lipocalin that is transcriptionall
8. 53 1 Version 140318 Tii Rat NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Human neutrophil gelatinase associated lipocalin NGAL also called human lipocalin 2 vas originally identified as an associated protein to 92 kDa human neutrophil type IV collagenase also called gelatinase B or matrix metalloproteinase 9 MMP 9 1 The rat ortholog neu related lipocalin NRL or a2 microglobulin related protein was identified as a protein highly overexpressed in mammary cancers The murine ortholog called 24p3 24 kDa superinducible protein Sip24 or uterocalin was identified as a protein induced in response to various proliferative signals and is highly expressed in uterine luminal fluids and epithelial cells Recently 24p3 has also been implicated in processes as diverse as apoptosis 2 and kidney cell differentiation 3 NGAL like most lipocalins is thought to modulate cellular processes by binding to ligand s and interacting with specific cell surface receptors Evidence for such a mammalian receptor for NGAL has recently been reported 2 One early hypothesis proposed that NGAL has immunomodulatory activity by binding and el aring lipophilic inflammatory mediators 4 such as the neutrophil tripeptide chemoattractant N formyl Met Leu Phe 5 6 NGAL is released from the secondary granules of activated neutrophils and plasma levels rise in i
9. Nephrol 12 787A Ohlsson S Wieslander J Segelmark M 2003 Increased circulating levels of proteinase ients with anti neutrophilic cytoplasmic autoantibodies associated systemic vasculitis in remission Clin Exp Immunol 131 528 535 Mishra J Ma Q Prada A Mitsnefes M Zahedi K Yang J Barasch n P 2003 Identification of neutrophil gelatinase associated lipocalin as a novel e i biomarker for ischemic renal injury J Am Soc Nephrol 14 2534 2543 Amin RP Vickers AE Sistare F Thompson KL Roman RJ Lawto Collins J Grissom S Bennett L Tucker CJ Wild S Kind C Oreff Naciff JM Cunningham M Tennant R Stevens J Car B Identification of putative gene based markers of renal toxi 465 479 Mishra J Mori K Ma Q Kelly C Barasch J Devaraja Neutrophil gelatinase associated lipocalin a novel early urinary biomarker for cisplatin nep xicity Am J Nephrol 24 307 315 Mishra J Dent C Tarabishi R Mitsnefes MM e Ruff SM Zahedi K Shao M Bean J Mori K Barasch J Devarajan P 2005 Neu gelatinase associated lipocalin NGAL as a biomarker for acute renal injury after cardiac sur Lancet 365 1231 1238 er J Hamadeh HK vis JW 2nd Curtiss S A Afshari CA 2004 iron Health Perspect 112 CY 8053 15 Version 140318 ii Rat NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Related Products CircuLex Rat NGAL ELISA Kit Cat CY 805
10. agulant If possible colle sma into a mixture of EDTA Na and Futhan5 to stabilize the sample against spontaneous vitro complement activation Immediately centrifuge samples at 4 C for 15 minutes at 1000 x g Assay immediately or store samples on ice for up to 6 hours before assaying Aliquots of may also be stored at below 70 C for extended periods of time Avoid repeated freeze thaw cycl Note Citrate plasma has not been validated for use in this assay Urine Immediately centrifuge samples at 4 C for 15 minutes at 15 000 supernatant or store on ice for up to 24 hours before assaying Aliquots below 70 C for extended periods of time Avoid repeated freeze tha Assay immediately the may also be stored at Other biological samples Remove any particulates by centrifug assay immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw c C CY 8053 7 Version 140318 Tii Rat NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex Rat NGAL Lipocalin 2 ELISA Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Since experimental conditions may vary an aliquot of the rat NGAL Standard within the kit should be included in each assay as a calibratof Disposable pipet
11. asuring absorbance in 96 well plates at dual wa s of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used n also be read at a single wavelength of 450 nm which will give a somewhat higher readi e Vortex mixer Qy e Microplate washer optional Manual washing is possible but not preferable f Software package facilitating data generation and analysis opti 500 or 1000 mL graduated cylinder e Reagent reservoirs e Deionized water of the highest quality e Disposable paper towels C CY 8053 5 Version 140318 Tii Rat NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations e Allow all the components to come to room temperature before use e All microplate strips that are not immediately required should be returned to the zip lock pouch Which must be carefully resealed to avoid moisture absorption e Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit e Rinse all detergent residue from glassware e Use deionized water of the highest quality e Do not mix reagents from different kits The buffers and reagents in this kit may contain preservatives or othetchemiu als Care should be taken to avoid direct contact with these reagents e Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the assa
12. nflammatory or infective conditions especially in bacterial infections 7 Thus the level of NGAL in plasma or serum has been proposed as a marker of infection However as levels of NGAL may also be raised in neoplastic conditions and renal disorders independently of any infective process this proposed application should be treated with caution An early and dramatic upregulation was later observed in Tat proximal tubule cells after ischemia reperfusion injury 8 and raised plasma levels of NGADjwere found to be strongly correlated with decreased renal function in patients with renal damage due to systemic vasculitis 9 The results for renal ischemia reperfusion injury were subsequently confirmed and extended to nephrotoxic agents 10 12 It has been suggested that urinary NGAL ley ls may Serve as an early marker for ischemic renal injury in children after cardiopulmonary bypass 13 Cat CY 8053 2 Version 140318 Tii Rat NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay The CycLex Research Product CircuLex Rat NGAL Lippocalin 2 ELISA Kit employs the quantitative sandwich enzyme immunoassay technique An antibody specific for rat NGAL has been pre coated onto a microplate Standards and samples are pipetted into the wells and the immobilized antibody binds any rat NGAL present After washing away any unbound substances a biotinylated antibod
13. ommercial use please contact us via email Cat CY 8053 16 Version 140318
14. s into the appropriate wells 4 Incubate the plate at room temperature ca 25 C fon 1 hour shaking at ca 300 rpm on an orbital microplate shaker 5 Wash 4 times by filling each well with Wash Buffery 350 uL using a squirt bottle multi channel pipette manifold dispenser or microplate washer 6 Add 100 uL of Biotinylated Detection Antibody into each well 7 Incubate the plate at room temperature ca25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker 8 Wash 4 times by filling each welk with Wash Buffer 350 uL using a squirt bottle multi channel pipette manifold dispenser onmaicroplate washer 9 Add 100 uL of HRP conjugated Streptavidin into each well 10 Incubate the plate at room temperature ca 25 C for 20 minutes shaking at ca 300 rpm on an orbital microplate shaker 11 Wash 4 times by filling each well with Wash Buffer 350 uL using a squirt bottle multi channel pipette manifold d spenser or microplate washer 12 Addd 00 uD of Substrate Reagent Avoid exposing the microtiter plate to direct sunlight Covering th plate with e g aluminum foil is recommended Return Substrate Reagent to 4 C immediately after the necessary volume is removed Cat CY 8053 9 Version 140318 Tii Rat NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 13 Incubate the plate at room temperature ca 25 C for 1
15. te tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r agents are supplied ready to use with the exception of 10X Wash Buffer and Rat NGAL Standard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of deionized distilled water ddH2O Mix well Store at 4 C for two weeks or lt 20 C for long term storage 2 Reconstitute Rat NGAL Standard with 1 0 mL of ddH O The concentration of the rat NGAL in vial should be 4 ng mL which is referred as a Master Standard of rat NGAL Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series below Mix each tube thoroughly before the next transfer The 1 000 pg mL standard Std 1 servesyas the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 150 uL of Master Standard 450 uL 1 000 pg mL Std 2 300 uL of Std 1 1 000 pg mL 300 uL 500 pg mL Std 3 300 uL of Std 2 500 pg mL 300 uL 250 pg mL Std 4 300 uL of Std 3 250 pg mL 300 uL 125 pg mL Std 5 300 uL of Std 4 125 pg mL 300 uL 62 5 pg mL Std 6 300 uL of Std 5 62 35 pg mL 300 uL 31 3 pg mL Std 7 300 uL of Std 6 313 pg mL 300 uL 15 6 pg mL Blank 300 uL 0 pg mL
16. terials Provided All samples and standards should be assayed in duplicate The following components are supplie are sufficient for the one 96 well microplate kit Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in a bag with a desiccant pack Wells are coated with anti rat NGAL antibody as a capture anti 10X Wash Buffer One bottle containing 100 mL of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle containing 50 mL of 1X buffer use for sample y use t Biotinylated Detection Antibody One bottle containing 12 mL of biotinylate Ready to use Rat NGAL Standard One vials containing 4 ng each of lyophilized recombinantira aa NGAL antibody HRP conjugated Streptavidin One bottle containing 12 mL conjugated streptavidin Ready to use orseradish peroxidase Substrate Reagent One bottle containing 20 mL of the chromogeni strate tetra methylbenzidine TMB Ready to use Stop Solution One bottle containing 20 mL of 1 N H SO4 Rea C CY 8053 4 Version 140318 Rat NGAL Lipocalin 2 ELISA Kit C ircuLex User s Manual 4 For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for sample preparation e Plate reader capable of me
17. ting the rat NGAL concentration Cat CY 8053 10 Version 140318 Tii Rat NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Measurement Range The measurement range is 15 6 pg mL to 1 000 pg mL Any sample reading higher than the highest standard should be diluted with Dilution Buffer in higher dilution and re assayed Dilution factors need to be taken into consideration in calculating the dog adiponectin concentration Troubleshooting paa The rat NGAL Standard should be run in duplicate 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 indicate insufficient washing If all instructions in the Detailed Protocol were followed accuratelygsuch results indicate a need for washer maintenance 3 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 Ada jSubstrate Reagent immediately after wash Reagent Stability All of the reagents included in the CycLex Research Product CircuLex Rat NGAL Lipocalin 2 ELISA Kit have been tested for stability Reagents shold nobbe used beyond the stated expiration date Upon receipt kit reagents should be stored at 4 C except
18. y or in argas where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containifg solutions in compliance with local regulations e Avoid contact with Substrate Solution which contains hydrogen peroxide e Avoid contact with Stop Solution which contains Sulfuric Acid e Wear gloves and eye protection when handling immunodiagnostic materials and samples of human origin and these reagents In case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medieal attention when necessary Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly e CAUTION Sulfuric Acid i a Strong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 8053 6 Version 140318 Rat NGAL Lipocalin 2 ELISA Kit CircuLex Ue Manual 4 For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Serum Use a serum separator tube and allow samples to clot for 60 30 minutes Centrifug samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or store sam 0 ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 C forme d periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA Na or heparin as the antico
19. y regulated by IL 3 deprivation Science 293 829 834 3 Yang J Goetz D Li J Y lt Wang W Mori K Setlik D Du T Erdjument Bromage H Tempst P Strong R and Barasch J 2002 Iron delivery pathway mediated by a lipocalin Mol Cell 10 1045 1056 4 Bundgaard J R Semgeloyy H Borregaard N and Kjeldsen L 1994 Molecular cloning and expression of a cDNA encoding NGAL a lipocalin expressed in human neutrophils BBRC 202 1468 1475 5 Chu S T Ling HJ tand Chen Y H 1997 Complex formation between a formyl peptide and 24p3 protein witha blocked N terminus of pyroglutamate J Pept Res 49 582 585 6 Sengelov H Boulay F Kjeldsen L and Borregaard N 1994 Subcellular localization and translocation Jof the receptor for N formylmethionyl leucyl phenylalanine in human neutrophils Biochem J 299 473 479 Cat CY 8053 14 Version 140318 Rat NGAL Lipocalin 2 ELISA Kit CircuLex User s Manual 10 11 12 13 Matthaeus T Schulze Lohoff E Ichimura T Weber M Andreucci M Park KM Alessan For Research Use Only Not for use in diagnostic procedures Xu SY Pauksen K Venge P 1995 Serum measurements of human neutrophil lipocalin discriminate between acute bacterial and viral infections Scand J Clin Lab Invest 55 125 131 Bonventre JV 2001 Co regulation of neutrophil gelatinase associated lipocalin metalloproteinase 9 in the postischemic rat kidney J Am Soc
20. y specific for rat NGAL is added to the wells followed by binding with horseradish peroxidase HRP 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 th addition of stopping reagent The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured at 450 nm The absorbance is proportional to the concentfation of rat NGAL A standard curve is constructed by plotting absorbance values versus rat NGAL concentrations of calibrators and concentrations of unknown samples are determined using this standard cilrve The CircuLex Rat NGAL Lipocalin 2 ELISA Kit is designed to measure the oncentration of rat NGAL Lipocalin 2 from serum plasma urine cultured macrophages or conditioned medium Summary of Procedure Add 100 uL of diluted sample to the wells 4 Incubate for 1 hourgat room temp Wash the wells Add 100 uL of biotinylated antitat NGAL antibody 4 Incubate for 1hour at room temp Wash the wells Add 100 uL of HRP conjugated Streptavidin 4 Incubate for 20 minutes at room temp Wash the wells t Add 400 DL oPSubstrate Reagent t Ad 100 uL of Stop Solution t Measure absorbance at 450 nm Cat CY 8053 3 Version 140318 Ti Rat NGAL Lipocalin 2 ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures L Ma
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