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1. b Positive Control c Sample 125 pl of Working Stock Item F 125 ul Prepared Sample a Standards 2 ml of Working Stock Item F 2 ml of Assay Diluent Perform a 2 fold dilution of i 100 ul of Working Working Stock Item F Stock Item F 100 ul Prepared Positive Control Final concentration 10 ng ml C Preparation of Standards 7 Label 6 microtubes with the following concentrations 1000 ng ml 100 ng ml 10ng ml 1 ng ml 100 pg ml and 0 pg ml Pipette 450 ul of biotinylated RBP4 Item F working solution prepared in step 6a into each tube except the 1 000 ng ml leave this one empty It is very important to make sure the concentration of biotinylated RBP4 is 10 ng ml in all standards 8 Briefly centrifuge the vial of RBP4 Standard Item C Pipette 8 ul of Item C and 792 ul of 10 ng ml biotinylated RBP4 working solution prepared in step 6a into the tube labeled 1000 ng ml Mix thoroughly This solution serves as the first standard 1 000 ng ml RBP4 standard 10 ng ml biotinylated RBP4 9 To make the 100 ng ml standard pipette 50 ul of the 1000 ng ml RBP4 standard into the tube labeled 100 ng ml Mix thoroughly 10 Repeat this step with each successive concentration preparing a dilution series as shown in the illustration below Each time use 450 ul of biotinylated RBP4 and 50 ul of the prior concentration until the 100 pg ml is reached Mix each tube thoroughly before the
2. XI Specificity This kit detects RBP4 183aa and all other active forms including RBP4 182 181 179 176 Cross Reactivity This EIA kit shows no cross reactivity with any of the cytokines tested Ghrelin Nesfatin Angiotensin Il NPY and APC XIV Publications Citing This Product 1 Anna P Lech C Jerzy J Robert F Assessment of serum IGF 1 and adipokines related to metabolic dysfunction in HIV infected adults Cytokine 64 2013 97 102 Species Human Sample Type Serum XIII Troubleshooting Guide e Check pipettes Poor standard e Inaccurate pipetting Briefly centrifuge Item C and dissolve curve e Improper standard dilution the powder thoroughly by gently mixing Briefly spin down vials before e Improper preparation of opening Dissolve the powder standard and or thoroughly biotinylated antibody Ensure sufficient incubation time e Too brief incubation times assay procedure step 2 may be done e Inadequate reagent overnight volumes or improper dilution Check pipettes and ensure correct preparation Low signal e Inaccurate pipetting Check pipettes e Air bubbles in wells e Remove bubbles in wells Review the manual for proper wash If using a plate washer ensure that all ports are unobstructed Make fresh wash buffer e Plate is insufficiently washed e Contaminated wash buffer Follow storage recomendations in e Improper storage of the sections IV and V Keep substrate ELISA kit solution protected from light
3. Invert the plate and blot it against clean paper towels Add 100 ul of each standard see Reagent Preparation Section C Positive Control see Reagent Preparation Section D and sample see Reagent Preparation Section E in appropriate wells Be sure to include a blank well Assay Diluent only Cover wells and incubate for 2 5 hours at room temperature with gentle shaking 1 2 cycles sec overnight or at 4 C Discard the solution and wash 4 times as directed in Step 3 Add 100 ul of prepared HRP Streptavidin solution see Reagent Preparation step 7 to each well Incubate for 45 minutes at room temperature with gentle 10 shaking It is recommended that incubation time should not be shorter or longer than 45 minutes Discard the solution and wash 4 times as directed in Step 3 Add 100 ul of TMB One Step Substrate Reagent Item H to each well Incubate for 30 minutes at room temperature in the dark with gentle shaking 1 2 cycles sec Add 50 ul of Stop Solution Item to each well Read at 450 nm immediately Assay Procedure Summary Prepare all reagents samples and standards as instructed Add 100 ul anti RBP4 to each well Incubate 1 5 hours at room temperature or overnight at 4 C Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or overnight at 4 C Add 100 ul prepared Streptavidin solution Incubate 45 minutes at room temperature Add 100 ul
4. e Stop solution Add stop solution to each well before reading plate RayBio ELISA Kits Over 2 000 ELISA kits available visit www RayBiotech com ELISA Kits html for details This product is for research use only 2015 RayBiotech Inc
5. R WD 8 9 10 11 Vil Additional Materials Required Microplate reader capable of measuring absorbance at 450 nm Precision pipettes to deliver 2 ul to 1 ml volumes Adjustable 1 25 ml pipettes for reagent preparation 100 ml and 1 liter graduated cylinders Absorbent paper Distilled or deionized water SigmaPlot software or other software which can perform four parameter logistic regression models Tubes to prepare standard or sample dilutions Orbital shaker Aluminum foil Plastic wrap Reagent Preparation Keep kit reagents on ice during reagent preparation steps Note Assay Diluent A should be used for dilution of samples Item F and Item C when testing plasma or serum samples 1X Assay Diluent B should be used for dilution of samples Item F and Item C when testing cell culture media or other sample types A Preparation of Plate and Anti RBP4 Antibody 1 2 Equilibrate plate to room temperature before opening the sealed pouch Label removable 8 well strips as appropriate for your experiment 5X Assay Diluent B Item E should be diluted 5 fold with deionized or distilled water Briefly centrifuge the anti RBP4 antibody vial Item N Then add 50 ul of 1X Assay Diluent B to the vial to prepare the antibody concentrate Pipette up and down to mix gently The antibody concentrate should then be diluted 100 fold with 1X Assay Diluent B This is your anti RBP4 antibody working
6. RayBio Human Mouse Rat RBP4 Enzyme Immunoassay Kit Catalog EIA RBP EIAM RBP EIAR RBP User Manual Last revised December 1 2015 Caution Extraordinarily useful information enclosed Re RayBiotech 1 ii The protein array pioneer ISO 13485 Certified 3607 Parkway Lane Suite 100 Norcross GA 30092 Tel 1 888 494 8555 Toll Free or 770 729 2992 Fax 770 206 2393 Web www RayBiotech com Email info raybiotech com Table of Contents ease SSSCS di i In oenemionsowin SSSCSCS d Cd m forne Cd ao paot ooo a ao Preparation A Preparation of Plate and Anti RBP4 Antibody B Preparation of Biotinylated Peptide Item F C Preparation of Standards D Preparation of Positive Control E Preparation of Samples F Preparation of Wash Buffer and HRP Strep vm Assay Procedure lege fix Assay Procedure Summary O Assay Procedure Summary pe Calculation of Results A Typical Data B Sensitivity a See eee C Detection Range D Reproducibility E Assay Diagram x Specifcty OOOO O O Specificity Ce xu Select Publications OO O O Select Publications a Troubleshooting Guide Please read the entire manual carefully before starting your experiment I Introduction RBP4 retinol binding protein 4 is an adipocyte secreted molecule that is elevated in the serum before the development of frank diabetes In obesity and type 2 diabetes expression of the GLUT4 glucose transporter is decreased selecti
7. TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature Add 50 ul Stop Solution to each well Read at 450 nm immediately X Calculation of Results Calculate the mean absorbance for each set of duplicate stands controls and samples and subtract the blank optical density Plot the standard curve using SigmaPlot software or other software which can perform four parameter logistic regression models with standard concentration on the x axis and percentage of absorbance see calculation below on the y axis Draw the best fit curve through the standard points Percentage absorbance B blank OD Bo blank OD where B OD of sample or standard and Bo OD of zero standard total binding A Typical Data These standard curves are for demonstration only A standard curve must be run with each assay RBP4 EIA 120 100 a oe S 80 4 S x 60 4 2 a io IK 20 4 S DON o D T T T T T 0 01 0 1 1 10 100 1000 10000 Peptide Cncentration ng ml B Sensitivity The minimum detectable concentrations of RBP4 is 460 pg ml C Detection Range 0 1 1 000 ng ml D Reproducibility Intra Assay CV lt 10 Inter Assay CV lt 15 E Assay Diagram Recommended Plate Layout Key Blank Buffer Only Total Binding Biotin RBP4 only Standard 1 1000 ng ml Standard 2 100 ng ml Standard 3 10 ng ml Standard 4 1 ng ml Standard 5 100 pg ml Pos Control Biotin with Item M
8. ated with Geis tae month at aor eee antibody Wash Buffer o A ft monthatas oe Item B 25 ml 25 mi of 20x concentrated soon 20X concentrated solution 1 month at 4 ft monthatas The first standard Standard RBP4 Peptide 2 vials of RBP4 Peptide 1 vial is enough to run 2 3 days at 4 C Item C each standard in duplicate Additional dilutions Do not store Anti RBP4 pee pee Item N 2 viais otaniRBPA of anti viais otaniRBPA 1 month ata at 4 month ata 30 ml contains 0 09 sodium azide as Assay Diluent A Item D preservative Diluent for standards and serum or plasma 15 ml of 5X concentrated buffer Diluent for Assay Diluent B Item E standards cell culture media or other sample 1 month at 4 C types and HRP Streptavidin ore RBP4 Peptide 2 vials of roe era RBP4 Peptide 1 vial is 2 3 es daysatae at es daysatae ore F roe era to assay the whole plate HRP oe 600 ee 400X concentrated HRP conjugated oe not store and Concentrate oe G ee oe Positive Control Item M Positive Control Item M Item M 1 1 vial of Positive Control of Positive 1 vial of Positive Control 2 3 at 2 3daysat4C TMB One Step Substrate 12 ml of 3 3 5 5 tetramethylbenzidine TMB i Reagent Item H buffer solution Stop Solution Item 1 8 8 ml of 0 2 M sulfuric acid of 0 2 M sulfuric acid E daea Return unused wells to the pouch containing desiccant pack reseal along entire edge Vi NOO
9. next transfer 90 nl 50l 90 ul 50 ul OS IS OAS OS SABE 1000 100 100 0 ng ml ng ml no sits pg ml pg ml D Positive Control Preparation 11 Briefly centrifuge the Positive Control vial Item M 12 Refer to step 6b This is a 2 fold dilution of the Positive Control The final concentration of biotinylated RBP4 should still be 10 ng ml The Positive Control is a cell culture media sample that serves as a system control to verify that the kit components are working The resulting OD will not be used in any calculations if no positive competition is observed please contact RayBiotech Technical Support The Positive Control may be diluted further if desired but be sure the final concentration of biotinylated RBP4 is 10 ng ml E Sample Preparation 13 If you wish to perform a 2 fold dilution of your sample proceed to step 6c If you wish to perform a higher dilution of your sample dilute your sample with the appropriate Assay Diluent before performing step 6c EXAMPLE to make a 4 fold dilution of sample a Dilute sample 2 fold 62 5 ul of sample 62 5 ul of the appropriate Assay Diluent b Perform step 6c 125 ul of working solution Item F 125 ul of sample prepared above The total volume is 250 ul enough for duplicate wells on the microplate It is very important to make sure the final concentration of the biotinylated RBP4 is 10 ng ml Note Optimal sample dilution factors should be determined empirically howeve
10. r you may reference below for recommended dilution factors for serum Human 4X Mouse 2X You may also contact technical support 888 494 8555 techsupport raybiotech com to obtain additional recommended dilution factors for serum F Preparation of Wash Buffer and HRP 14 15 16 17 If Item B 20X Wash Concentrate contains visible crystals warm to room temperature and mix gently until dissolved Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1X Wash Buffer Briefly centrifuge the HRP Streptavidin vial Item G before use Dilute the HRP Streptavidin concentrate 400 fold with 1X Assay Diluent B Note do not use Assay Diluent A for HRP Streptavidin preparation in step 17 VIII Assay Procedure Keep kit reagents on ice during reagent preparation steps It is recommended that all standards and samples be run at least in duplicate Add 100 ul of Anti RBP4 Antibody Item N See Reagent Preparation step 3 to each well Incubate for 1 5 hours at room temperature with gentle shaking 1 2 cycle sec You may also incubate overnight at 4 C Discard the solution and wash wells 4 times with 1X Wash Solution Buffer 200 300 ul each Washing may be done with a multichannel pipette or an automated plate washer Complete removal of liquid at each step is essential to good assay performance After the last wash remove any remaining Wash Buffer by aspirating or decanting
11. s and standards are then added to the plate where the biotinylated RBP4 peptide competes with endogenous unlabeled RBP4 for binding to the anti RBP4 antibody After a wash step any bound biotinylated RBP4 then interacts with horseradish peroxidase HRP streptavidin which catalyzes a color development reaction The intensity of the colorimetric signal is directly proportional to the amount of captured biotinylated RBP4 peptide and inversely proportional to the amount of endogenous RBP4 in the standard or samples A standard curve of known concentration of RBP4 peptide can be established and the concentration of RBP4 peptide in the samples can be calculated accordingly ll How It Works Anti lgG antibody Bi ae Y Y pre coated on the plate Y Y Target molecule Biotinylated ry in sample Peptide w tk gt Capture antibody p pee is added to the wells Biotin peptide Standard w A Sample interact competitivly Ma a i for spots on the capture antibodies T Y on a i el Add HRP Streptavidin and Color Substrate Data Analysis IV Storage The entire kit may be stored at 20 C to 80 C for up to 6 months from the date of shipment For extended storage it is recommended to store at 80 C Avoid repeated freeze thaw cycles For prepared reagent storage see table below V Reagents Size Description Storage Sabiny Stability RBP4 EPA microplate tem A Item A PBPa microplate tem a wells 12 ee x 8 wells co
12. solution which will be used in step 2 of Assay Procedure Section VIII 6 Note The following steps may be done during the antibody incubation procedure step 2 of Assay Procedure B Preparation of Biotinylated RBP4 Item F 5 Briefly centrifuge the vial of Biotinylated RBP4 Item F before use 6 See the image below for proper preparation of Item F Transfer the entire contents of the Item F vial into a tube containing 10 ml of the appropriate Assay Diluent This is your Working Stock of Item F Pipette up and down to mix gently The final concentration of biotinylated RBP4 will be 20 ng ml a Second Dilution of Item F for Standards Add 2 ml of Working Stock Item F to 2 ml of the appropriate Assay Diluent The final concentration of biotinylated RBP4 will be 10 ng ml b Second Dilution of Item F for Positive Control Add 100 ul of Working Stock Item F to 100 ul of the prepared Positive Control Item M See section D for Positive Control preparation The final concentration of biotinylated RBP4 will be 10 ng ml c Second Dilution of Item F for samples Add 125 ul of Working Stock Item F to 125 ul of prepared sample see section E for sample preparation This is a 2 fold dilution of your sample The final concentration of biotinylated RBP4 will be 10 ng ml 1 vialis enough to run the itemF whole plate First Dilution Add entire vial of Item F to Working Stock Item F 10 ml Assay Diluent Second Dilution 5
13. vely in adipocytes Adipose specific Glut4 knockout mice show insulin resistance secondarily in muscle and liver Recent studies have shown that serum RBP4 levels are elevated in insulin resistant mice and humans with obesity and type 2 diabetes RBP4 levels are normalized by rosiglitazone an insulin sensitizing drug Transgenic overexpression of human RBP4 or injection of recombinant RBP4 in normal mice causes insulin resistance Conversely genetic deletion of RBP4 enhances insulin sensitivity Fenretinide a synthetic retinoid that increases urinary excretion of RBP4 normalizes serum RBP4 levels and improves insulin resistance and glucose intolerance in mice with obesity induced by a high fat diet Increasing serum RBP4 induces hepatic expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase PEPCK and impairs insulin signalling in muscle Thus RBP4 is an adipocyte derived signal that may contribute to the pathogenesis of type 2 diabetes Lowering RBP4 could be a new strategy for treating type 2 diabetes Thus RBP4 appears to identify insulin resistance and associated cardiovascular risk factors in subjects with varied clinical presentations ll General Description The RayBio RBP4 Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting RBP4 peptide based on the competitive enzyme immunoassay principle In this assay a biotinylated RBP4 peptide is spiked into the samples and standards The sample

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