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1. C 60 min 300 rom Wash x3 350 uL x 3 Substrate Solution 100 uL Color Development 25 C 10 20 min 300 rpm Stop Solution 100 uL Read Absorbance at 450 nm B Bridge International Inc 10 www b bridge com Rat Resistin ELISA Kit User Manual Vil Calculation of Results 1 Subtract the mean absorbance value of the 0 ng ml blank from each mean absorbance value of the standard series and samples tested Net Absorbance 2 Plot the concentrations of each standard and the calculated Net Absorbances on the X axis and Y axis respectively Fit an appropriate regression curve to the points 3 Determine the resistin concentrations of the samples by interpolation of the regression curve Figure 3 Typical Standard Curve Rat Resistin ELISA Standard Curve 3 00 2 50 2 00 1 50 E o Te Q O 1 00 0 50 0 00 0 1 1 10 Concentration of Rat Resistin ng ml Figure 3 Standard Curve for rat resistin is plotted using the four parameter function as a proportion of rat resistin concentration and absorbance at 450 nm B Bridge International Inc www b bridge com Rat Resistin ELISA Kit User Manual Vill Troubleshooting Guide 1 Lack of signal or weak signal in all wells Possible explanations e Omission of a reagent or a step e Improper preparation or storage of a reagent e Assay performed before reagents were allowed to come to 25 C e Plate reader did not perform we2. Overview Resistin a product of the RSTN gene is a peptide hormone belonging to the class of cysteine rich secreted proteins which is termed the RELM family and is also described as ADSF Adipose Tissue Specific Secretory Factor and FIZZ3 Found in Inflammatory Zone Rat resistin contains 114 amino acids as a prepeptide and its hydrophobic signal peptide is cleaved before secretion Resistin circulates in rat blood as a dimeric protein consisting of two 96 amino acid polypeptides which are disulfide linked Resistin may be an important link between obesity and insulin resistance Mouse resistin specifically produced and secreted by adipocyte acts on skeletal muscle myocytes hepatocytes and adipocytes to reduce their sensitivity to insulin Steppan et al have suggested that resistin suppresses the ability of insulin to stimulate glucose uptake They have also suggested that resistin is present at elevated levels in the blood of obese mice and is down regulated by fasting and antidiabetic drugs TZDs On the other hand several studies demonstrated reduced resistin expression in adipose tissue of obese mice and increased levels in leptin deficient ob ob mice and Zucker diabetic fatty rats in response to TZDs Other studies have shown that mouse resistin increases during the differentiation of adipocytes but it also seems to inhibit adipogenesis Nevertheless the human adipogenic differentiation is likely to be associated with a down regulat
3. washer is used the plate frame should be completely filled with wells by adding as many null strips as necessary Identify well position s for each sample on a data sheet or plate map Add 100 ul of diluted Standards Quality Controls and samples to the appropriate number of antibody coated wells Every plate must include the standard series to properly correlate the sample readings Cover plate s securely with a plate sealer and incubate at room temperature 25 C for 60 minutes on an orbital microplate shaker at ca 300 rpm After incubation thoroughly wash the plate s 3 times with Wash Solution as follows a Carefully remove the plate sealer avoiding splashing and discard appropriately b Completely aspirate the liquid from the wells using a plate washer c Fill each well with 1X Wash Solution 350 ul well and immediately aspirate Avoid overflow d Repeat Step 4c two more times for a total of three washes e Invert the plate s and gently tap on a clean absorbent towel Dispense 100 ul of the Biotinylated Secondary Antibody Solution into each well Cover plate s securely with a new clean plate sealer and incubate at room temperature 25 C for 60 minutes on an orbital microplate shaker at ca 300 rpm Repeat the wash procedure described in step 4 Dispense 100 ul of Streptavidin HRP Conjugate Solution into each well Cover plate s securely with a plate sealer and incubate at room temperature 25 C for 30 minutes on an
4. B Bridge International Inc wA Rat Resistin ELISA Kit User Manual UM 101401 Published 01 June 2005 Catalog K1014 1 See List of Components for Storage Conditions FOR RESEARCH USE ONLY Rat Resistin ELISA Kit User Manual Table of Contents l Introduction and Protocol Overview 3 Il List of Components 5 III Additional Materials Required 6 IV Reagent Preparation and Storage 7 V Sample Preparation and Storage 8 VI Rat Resistin ELISA Protocol 9 VII Calculation of Results 11 VIII Troubleshooting Guide 12 IX References 13 Notice to Purchaser This product is to be used for Research Purposes Only It is not to be used for Drug or Diagnostic Purposes nor is it intended for Human Use B Bridge products may not be resold modified for resale or used to manufacture commercial products without the express written consent of B Bridge International Inc EXCEPT AS OTHERWISE EXPRESSLY SET FORTH IN THIS USER MANUAL B BRIDGE DOES NOT MAKE ANY REPRESENTATION OR WARRANTIES OR CONDITIONS OF ANY KIND EITHER EXPRESSED OR IMPLIED WITH RESPECT TO THE PRODUCTS OR INFORMATION DISCLOSED HEREUNDER INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FIT FOR A PARTICULAR PURPOSE OR NONINFRINGEMENT OF THE INTELLECTUAL PROPERTY RIGHTS OF THIRD PARTIES 2004 B Bridge International Inc All Rights Reserved B Bridge International Inc www b bridge com Rat Resistin ELISA Kit User Manual l Introduction and Protocol
5. agano C Federspil G Vettor R Resistin and adiponectin expression in visceral fat of obese rats effect of weight loss Obes Res 10 11 1095 103 2002 Nogueiras R Gallego R Gualillo O Caminos J E Garcia Caballero T Casanueva F F Dieguez C Resistin is expressed in different rat tissues and is regulated in a tissue and gender specific manner FEBS Lett 31 548 1 3 21 7 2003 Pravenec M Kazdova L Landa V Zidek V Mlejnek P Jansa P Wang J Qi N Kurtz TW Transgenic and recombinant resistin impair skeletal muscle glucose metabolism in the spontaneously hypertensive rat J Biol Chem 278 45209 45215 2003 Rajala M W Obici S Scherer P E Rossetti L Adipose derived resistin and gut derived resistin like molecule beta selectively impair insulin action on glucose production J Clin Invest 111 2 225 30 2003 Satoh H Nguyen M T Miles P D Imamura T Usui l Olefsky J M Adenovirus mediated chronic hyper resistinemia leads to in vivo insulin resistance in normal rats J Clin Invest 114 2 224 31 2004 Steppan C M Lazar M A The current biology of resistin Journal of Internal Medicine 255 439 447 2004 Steppan C M Lazar M A Resistin and obesity associated insulin resistance TRENDS in Endocrinology and Metabolism 13 1 18 23 2002 Steppan C M at al A Family of Tissue specific Resistin like Molecules Proc Natl Acad Sci USA 98 2 502 506 2001 Steppan C M et al The Hor
6. ion of resistin gene expression The B Bridge Rat Resistin ELISA Kit is designed to measure the concentration of rat resistin in rat serum or tissue culture medium B Bridge International Inc www b bridge com Rat Resistin ELISA Kit User Manual The principle of the assay is shown in Figure 1 Standards or samples are incubated in microtiter wells coated with a rabbit polyclonal anti rat resistin antibody The wells are washed and biotin labelled rabbit polyclonal anti rat resistin antibody is added and incubated with the captured resistin After a thorough wash streptavidin horseradish peroxidase HRP conjugate is added Following subsequent incubation and washing the bound conjugate is allowed to react with the substrate H2O2 tetramethylbenzidine TMB The reaction is quenched by addition of acidic solution and absorbance of the resulting product is measured at 450 nm The absorbance correlates to the concentration of resistin A standard curve is constructed by plotting absorbance values versus known resistin standard concentrations and concentrations of unknown samples are determined by interpolation on this curve Figure 1 Assay Principle Biotin labeled rabbit ee polyclonal anti rat Streptavidin HRP Resistin resistin antibody Conjugate Se 2a 1st Reaction 2nd Reaction 3rd Reaction Color Development gt gt gt gt OOO 090 Saan A rabbit polyclonal anti rat resistin antibody coated plate B Bridge Internatio
7. ity Controls are now ready to use Reconstituted Quality Controls are stable until the expiration date at 20 C Note Do not mix reagents from different kits unless they have the same lot number B Bridge International Inc 7 www b bridge com Rat Resistin ELISA Kit User Manual V Sample Preparation and Storage Allow all the reagents to equilibrate at room temperature 25 C prior to the start of the sample preparation 1 ForAssaying Samples in singlets Mix 10 ul of samples with 190 ul of 1X Sample Diluent 2 For Assaying Samples in duplicates Mix 15 ul of samples with 285 ul of 1X Sample Diluent Note Serum samples should be stored frozen preferably at 80 C Repeated freeze thawing should be avoided Undiluted samples are stable at least a week at 2 8 C or one day at room temperature 25 C Diluted samples have to be stored frozen B Bridge International Inc 8 www b bridge com Rat Resistin ELISA Kit User Manual VI Rat Resistin ELISA Protocol Note Allow all reagents to come to room temperature 25 C prior to the start of the assay and prepare 1X Wash Solution Quality Controls Resistin Standards and Samples as described in the previous sections 1 10 11 12 13 14 B Bridge International Inc Remove Primary Antibody Coated Plate from its foil pouch Remove any unneeded strips from the plate frame reseal them in the foil pouch and return the foil pouch to 2 8 C If a 96 well plate
8. ll 2 High signal and background in all wells Possible explanations e Improper or inadequate washing be certain that all wash volumes and repetitions were correct e Improper dilution of detection antibody e Overdeveloping decrease the incubation time before the Stop Solution is added 3 High background in sample wells only Possible explanations e Sample concentration was too high e Improper dilution of detection antibody 4 Weak signal in sample wells only Possible explanations e Sample concentration was too low e Improper dilution of detection antibody B Bridge International Inc 42 www b bridge com Rat Resistin ELISA Kit User Manual IX References Banerjee R R Lazar M A Dimerization of resistin and resistin like molecules is determined by a single cysteine The Journal of Biological Chemistry 276 25970 25973 2001 Gong H Ni Y Guo X Fei L Pan X Guo M Chen R Resistin promotes 3T3 L1 preadipocyte differentiation Eur J Endocrinol 150 6 885 92 2004 Juan CC at al Suppressed Gene Expression of Adipocyte Resistin in an Insulin resistant Rat Model Probably by Elevated Free Fatty Acids Biochemical and Biophysical Research Communications 289 1328 1333 2001 Kim K H et al A Cysteine rich Adipose Tissue specific Secretory Factors Inhibits Adipocyte Differentiation The Journal of Biological Chemistry 276 14 11252 11256 2001 Milan G Granzotto M Scarda A Calcagno A P
9. m optional Orbital microplate shaker capable of approximately 300 rom Well closed containers such as microtubes 1 5 ml or more in capacity www b bridge com Rat Resistin ELISA Kit User Manual IV Reagent Preparation and Storage Allow all the reagents to equilibrate at room temperature 25 C prior to the start of the reagent preparation 1 1X Wash Solution Prepare 1X Wash Solution by mixing all of the 10X Wash Solution 100 ml with 900 ml of deionized water or equivalent After preparation store 1X Wash Solution at 2 8 C The diluted Wash Solution is stable for 1 month at 2 8 C 2 Rat Resistin Master Standard Solution Prepare each Resistin Standard by diluting the supplied resistin standard solutions 20 ng ml with 1 ml Sample Diluent for duplicates Do not store the diluted standard solutions The standards are stable until the expiration date at 20 C Table 1 Serial Dilution of Resistin Standards Master Standard Vol 1X Diluent Concentration Master Sid 0 ul 20 ng ml 500 ul of 20 ng ml Master Std 500 ul 10 ng ml 500 ul of 10 ng ml Std 500 ul 5 ng ml 500 ul of 5 ng ml Std 500 ul 2 5 ng ml 500 ul of 2 5 ng ml Std 750 ul 1 ng ml 500 ul of 1 ng ml Std 500 ul 0 5 ng ml 500 ul of 0 5 ng ml Std 500 ul 0 25 ng ml 3 Quality Controls Reconstitute the Quality Controls by adding 350 ul of sample diluent to each vial containing lyophilized Quality Controls Reconstituted Qual
10. mone Links Obesity to Diabetes Nature 409 307 312 2001 Way J M et al Adipose Tissue Resistin Expression Is Severely Suppressed in Obesity and Stimulated by Peroxisome Proliferator activated Receptor y Agonists The Journal of Biological Chemistry 276 28 25651 25653 2001 B Bridge International Inc www b bridge com
11. nal Inc www b bridge com Rat Resistin ELISA Kit User Manual ll List of Components e Store all components at 2 8 C DO NOT FREEZE 1 10X WASH SOLUTION 1 Bottle 100 ml 2 1X SAMPLE DILUENT 2 Bottle 10 ml 3 PRIMARY ANTIBODY COATED PLATE 1 Plate One plate holds 12x8 well strips 96 wells with adsorbed Rabbit Polyclonal Anti Rat Resistin Antibody Plate is provided in a resealable foil pouch with desiccant 4 RAT RESISTIN MASTER STANDARD 1 Vial lyophilized 20 ng ml 5 QUALITY CONTROL 2 Vial lyophilized High and Low 6 BIOTINYLATED SECONDARY ANTIBODY SOLUTION 1 Bottle 13 ml Rabbit Polyclonal Anti Rat Resistin Antibody Biotin Labeled 7 CONJUGATE SOLUTION 1 Bottle 13 ml Streptavidin Horseradish Peroxidase Conjugate 8 SUBSTRATE 1 Bottle 13 ml H202 Tetramethylbenzidine TMB 9 STOP SOLUTION 1 Bottle 13 ml 0 2M H2SO PLATE SEALERS 1 Package Six sealers per package MSDS forms are available on our website please visit www b bridge com B Bridge International Inc www b bridge com Rat Resistin ELISA Kit User Manual Additional Materials Required The following materials are required but not supplied B Bridge International Inc Graduated cylinder Micropipettor s and disposable pipette tips Null strips for 96 well plate 96 well plate or manual strip washer Paper towels or absorbent paper Plate reader capable of measuring absorbance at a wavelength of 450 nm reference filter at 620 650 n
12. orbital microplate shaker at ca 300 rpm Repeat the wash procedure described in step 4 Dispense 100 ul of Substrate Solution into each well Avoid exposing the microtiter plate to direct sunlight Covering the plate with aluminum foil is recommended Cover plate s securely with a plate sealer and incubate at room temperature 25 C for 10 to 20 minutes on an orbital microplate shaker at ca 300 rpm Dispense 100 ul of Stop Solution into each well The plate should be read immediately Read the plate at 450 nm using a plate reader If using a dual filter instrument the recommended reference wavelength is 620 650 nm www b bridge com Rat Resistin ELISA Kit User Manual Note If the microplate reader is not capable of reading absorbance greater than the absorbance of the highest standard the standard curve constructed using the values measured at 405 nm can be used to determine resistin concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Figure 2 Flow Chart of Assay Procedure A Rabbit polyclonal Anti Rat Resistin Polyclonal Antibody coated plate Diluted Standards Quality Controls and Samples 100 uL 1st Reaction 25 C 60 min 300 ri Prepare Wash Solution ARA ae ee Wash x3 350 uL x 3 Biotinylated Rabbit Polyclonal Anti Rat Resistin Antibody Solution 100 uL 2nd Reaction 25 C 60min 300rpm Streptavidin HRP Conjugate Solution 100 uL 3rd Reaction 25
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