Manual - RayBiotech, Inc.
Contents
1. Item A CART microplate toma wells 12 ee x 8 wells coated with Geis tae month at aor a antibody Wash Buffer oe 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 CART Peptide 2 vials of CART 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 CART oe oe Item N 2 Bvialsofanticant of anti Bvialsofanticant 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 a CART Peptide 2 vials of oe CART Peptide 1 vial is 2 3 es daysatae at es daysatae a F oe to assay the whole plate HRP fe 600 eee 80X concentrated HRP conjugated oe not store and Concentrate fe G eee 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 ET daea Return unused wells to the pouch containing desicc2. Incubate 45 minutes at room temperature Add 100 ul 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 CART EIA 120 100 ee 60 BiBO 40 4 T T T T T T 0 001 0 01 0 1 1 10 100 1000 10000 Peptide Concentration pg ml B Sensitivity The minimum detectable concentrations of CART is 13 5 pg ml C Detection Range 1 10 000 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 CART only Standard 1 10 000 pg ml Standard 2 1000 pg ml Standard 3 100 pg ml Standard 4 10 pg ml Standard 5 1
3. is widely expressed in the central nervous system and the periphery but is particularly concentrated in the hypothalamus CART peptides appear to have an important function in the regulation of energy homeostasis CART interacts with a number of central appetite circuits Hypothalamic CART expression is regulated by a number of peripheral factors including the adipose hormone leptin Several research groups have shown that intracerebroventricular administration of CART 55 102 reduces appetite and stimulates energy expenditure Animal studies show that CART knockout mice demonstrate small but significant increases in weekly food consumption body weight and fat mass compared with their wild type littermates when on a high fat diet These reports suggest that CART peptide acts as an endogenous inhibitor of food intake On the other hand hypothalamic CART may also play an orexigenic role under specific circumstances as injection of CART 55 102 into specific hypothalamic nuclei increases food intake The CART system demonstrates multiple roles in energy homeostasis depending on the internal and external milieu The development of specific CART antagonists would be very useful in dissecting these multiple roles ll General Description The RayBio CART Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting CART peptide based on the competitive enzyme immunoassay principle In this assay a biotinylated CART peptide i
4. pg ml Pos Control Biotin with Item M XI Specificity 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 Hehir MP Laursen H Higgins MF Brennan DJ O Connor DP McAullife FM Maternal and fetal cocaine and amphetamine regulated transcript in diabetic and non diabetic pregnancy Gynecol Endocrinol 2012 28 9 682A A 685 Species Human Sample Type Serum 2 Wysokinski A et al Blood serum levels of CART peptide in patients with schizophrenia Psychiatry Research 2014 August epub ahead of print DOI 10 1016 j psychres 2014 08 017 on clozapinemonotherapy 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
5. remove any remaining Wash Buffer by aspirating or decanting 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 CART 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
6. RayBio Human Mouse Rat CART Enzyme Immunoassay Kit Catalog EIA CART EIAM CART EIAR CART 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 CART 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 Cocaine and amphetamine regulated transcript CART codes for a neuropeptide system with a number of biological roles The high conservation of CART across species suggests that it has an important role in mammalian physiology CART
7. ant pack reseal along entire edge Vi NOOR 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 CART 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 CART 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 Di
8. hed Mix each tube thoroughly before the next transfer 50 ul 50 ul 50 ul 50 ul ES ISOS ss 255556 10000 1000 100 pg ml pg ml pg ml pg ml 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 CART should still be 100 pg 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 CART is 100 pg 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 CART is 100 pg ml Note Optimal sample dil
9. luent B This is your anti CART antibody working 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 CART Item F 5 Briefly centrifuge the vial of Biotinylated CART 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 CART will be 200 pg 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 CART will be 100 pg 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 CART will be 100 pg 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 CART will be 100 pg ml 1 vial is enough to run the whole plate Negi First Dilution Working Stock Item F Add entire vial of Item F t
10. o 10 ml Assay Diluent Second Dilution b Positive Control a Sample 125 ul 100 ul of Working of Working Stock Stock Item F 100 ul Item F 125 ul Prepared Positive Prepared Sample Control a Standards 2 ml of Working Stock Item F 2 ml of Assay Diluent Perform a 2 fold dilution of Working Stock Item F Final concentration 100 pg ml C Preparation of Standards 7 Label 6 microtubes with the following concentrations 10 000 pg ml 1 000 pg ml 100 pg ml 10 pg ml 1 pg ml and 0 pg ml Pipette 450 ul of biotinylated CART Item F working solution prepared in step 6a into each tube except the 1 000 pg ml leave this one empty It is very important to make sure the concentration of biotinylated CART is 100 pg ml in all standards 8 Briefly centrifuge the vial of CART Standard Item C Pipette 8 ul of Item C and 792 ul of 100 pg ml biotinylated CART working solution prepared in step 6a into the tube labeled 10 000 pg ml Mix thoroughly This solution serves as the first standard 10 000 pg ml CART standard 100 pg ml biotinylated CART 9 To make the 1 000 pg ml standard pipette 50 ul of the 10 000 pg ml CART standard into the tube labeled 1 000 pg 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 CART and 50 ul of the prior concentration until the 1 pg ml is reac
11. 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 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
12. s spiked into the samples and standards The samples and standards are then added to the plate where the biotinylated CART peptide competes with endogenous unlabeled CART for binding to the anti CART antibody After a wash step any bound biotinylated CART 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 CART peptide and inversely proportional to the amount of endogenous CART in the standard or samples A standard curve of known concentration of CART peptide can be established and the concentration of CART peptide in the samples can be calculated accordingly ll How It Works Anti lgG antibody i ae Y Y pre coated on the plate Y Y Target molecule Biotinylated ry in sample Peptide t gt Capture antibody we is added to the wells Biotin peptide Standard w A Sample interact competitivly Ma a i for spots on the capture antibodies T Y BNP EIA Kit il 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 CART CART Microplate om A
13. ution factors should be determined empirically however you may reference below for recommended dilution factors for serum Human 4X Mouse 4X Rat 2X If you have any questions regarding the recommendended dilutions you may contact technical support at 888 494 8555 or techsupport raybiotech com 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 80 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 CART 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
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