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    Home > Active Ingredient News > Drugs Articles > Rat TfR ELISA Kit

    Rat TfR ELISA Kit

    • Last Update: 2010-01-11
    • Source: Internet
    • Author: User
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    Product No.: eia06720 read the manual thoroughly before use This ELISA kit is based on the principle of classical enzyme-linked sandwich technology, which can measure TfR of rats, and can only be used for research purposes, not for medical diagnosis Working principle: TfR transferrin receptor is a kind of transmembrane glycoprotein linked by non disulfide bond The protein consists of two identical peptide chains, each with a molecular weight of 95kd TfR transferrin iron complex enters the cell fluid from the cell membrane through the internalization of endocytic vesicles Iron remains in the cell fluid and the TfR- transferrin complex is recycled back to the cell surface The provided ELISA kit is a typical sandwich ELISA kit The prepackaged antibody is polyclonal antibody The antibody in the detection phase was also polyclonal and labeled with biotin The sample and biotin labeled antibody were added to the enzyme plate pore successively and then washed by PBS or TBS After that, we added the peroxidase labeled avidin reaction, washed PBS or TBS thoroughly, and then developed the color with the substrate TMB TMB was converted to blue under the catalysis of peroxidase, and to Zui yellow under the action of acid There was a positive correlation between the color depth and the factors to be measured Content of each kit (96T) material quantity standard 96T (2vial) pre coated 96 pore plate 96T (8 × 12) biotin labeled antibody 96T (1:100) ABC (avidin peroxidase complex) 96T (1:100) sample diluent 96T × 2 antibody diluent 96T × 1 ABC diluent 96T × 1 TMB developer a 96T × 1 TMB developer B 96T × 1 TMB termination solution 96T × 1 1.37 ℃ incubator for reagents and equipment not provided but needed for ELISA special detergent 96T × 1 (1:25) 2 Standard Specification enzyme labeling instrument 3 Automatic washing machine 4 Single channel or multi-channel adjustable pipette and its suction head 5 Distilled water, volumetric flask, etc 6 Clean test tube and eppendof tube Note 1 For the kit taken from - 20 ℃, Zui should be thawed at 4 ℃ overnight Each bottle of thawed solution in the box shall be gently shaken before opening the bottle cap to avoid stratification during thawing, otherwise the concentration will be uneven 2 Balance at room temperature for at least 30 minutes before opening the kit 3 When preparing various reagents, remember to mix them well! 4 When using the kit for the first time, the user should centrifuge all kinds of reagent tubes for several minutes so that the reagent can be concentrated at the bottom of the tube 5 It is recommended that all standards and samples be tested in duplicate 6 Strictly avoid the drying of enzyme plate during operation Drying will make the biological components on the enzyme plate inactivate quickly! 7 To avoid cross contamination, avoid reusing the suction head and test tube in your hand Washing method: manually wash the board: suck (do not touch the board wall) or shake off the liquid in the enzyme standard board; lay several layers of absorbent paper on the experimental platform, and pat the enzyme standard board downward for several times; inject at least 0.35ml of recommended PBS or TBS washing buffer into the hole and soak for 1-2 minutes Repeat the process as many times as necessary Automatic plate washing: if there is an automatic plate washing machine, it shall be used in the formal experiment process after skilled use If the preparation and preservation of samples are not analyzed immediately, they shall be refrigerated and preserved after sub packaging, and repeated freezing and thawing shall be avoided Serum: collect the blood with a clean tube, coagulate at room temperature for 2 hours or overnight at 4 ℃, centrifugate 1000 × g for 10 minutes, and collect the serum Immediately analyze or repack and freeze at - 20 ℃ Cell culture, tissue homogenate, body fluid - remove the precipitate by centrifugation, analyze it immediately or freeze it at - 20 ℃ after repacking The general principle of sample dilution is that the user should consult the literature to understand the content of the factors to be measured in the sample, and determine the appropriate dilution ratio, so that the concentration of the factors to be measured in the diluted sample is within the Zui optimal detection range of the ELISA kit The dilution of samples shall be recorded in detail Preparation and preservation of reagents A dilution and use of standards: prepare within 2 hours before use Add 1ml of sample diluent into the lyophilized tube, dissolve it thoroughly, and then dilute it by multiple ratio The diluted standard should be used within 2 hours B Biotin labeled antibody working solution: prepare within 2 hours before use 1 Calculate the total dosage according to the requirement of 0.1ml per hole (0.1-0.2ml more should be prepared in actual preparation) 2 Prepare the working solution according to the ratio of 10ul biotin labeled antibody and 990ul antibody diluent Mix gently C Preparation of ABC working solution: prepare within 1 hour before use 1 Calculate the total dosage according to the requirement of 0.1ml for each hole (0.1-0.2ml more should be prepared in actual preparation) 2 The working solution was prepared according to the ratio of 10ul avidin peroxidase complex (ABC) and 990ul ABC diluent Mix gently D Preparation of TMB working solution: 30 minutes before use, prepare TMB working solution according to the proportion of 9 parts of TMB developing solution a and 1 part of TMB developing solution B Operation procedure 1 Determine the number of enzyme plate holes coated with antibody required for this test 2 Add 0.1ml of diluted standard into a row of 7 holes in turn, and only add the sample diluent into one hole as the control The treated samples were added as 100ul per hole 3 Enzyme plate and cover, reaction at 37 ℃ for 90 minutes 4 After reaction, use the automatic washing machine to absorb the liquid in the enzyme standard plate; or shake off the liquid in the enzyme standard plate, and then pat it on the absorbent paper for several times Wash twice 5 Add the prepared biotin antibody working solution in order of 0.1ml per pore Reaction at 37 ℃ for 60 minutes Wash 6.0.01m TBS or 0.01M PBS for 3 times and soak for about 1 minute each time 7 Add 0.1ml ABC working solution into each hole in turn 37 ℃ for 30 minutes Wash 8.0.01m TBS or 0.01M PBS for 5 times and soak for about 1-2 minutes each time 9 Add 0.1ml of TMB working solution to each hole in turn, and avoid light reaction at 37 ℃ During the reaction process, observe frequently When the first 3-4 holes of the standard are obviously blue gradient, and the difference between the last 3-4 holes is not obvious, add 0.1ml/hole of TMB termination solution (the color reaction Zui should not be longer than 30 minutes) 10 The O.D value was measured at 450 nm by enzyme labeling instrument 11 Find out the corresponding concentration on the coordinate according to the absorption value of the sample Users can also use a variety of applications to calculate It should be remembered that as the sample is diluted by N times, its actual concentration should be x n Summary of operation procedures: prepare reagents, add samples and standards to the prepared samples and standards, wash the plate twice in 90 minutes at 37 ℃, add biotinylated antibody working solution, wash the plate three times in 60 minutes at 37 ℃, add ABC working solution, wash the plate five times in 30 minutes at 37 ℃, add TMB developer, add TMB termination solution at 37 ℃ and read OD value within 30 minutes The detection range of the factors to be determined was 100 nmol / ml → 1.5 nmol / ml Sensitivity: < 0.5 nmol / ml specificity: no cross reaction between the system and other factors Storage: - 20 ℃ [4 ℃ for a short period (such as two weeks)] validity period: 12 months (- 20 ℃) Purpose: for quantitative analysis of liquid samples in vitro REFERENCES 1 Nature 388:394-397(1997) 2 Nat Genet 25:187-191(2000) 3 Protein Sci 13:2819-2824(2004) 4 Proc Natl Acad Sci U.S.A 95:588-593(1998) 5 Nature 408:111-115(2000) 6 J Biol Chem 277:1845-1854(2002) 7 J Immunol 173:3589-3593(2004) 8 Cell 130:906-917(2007) 9 Genetics 158:1657-1664(2001) 10 Hum Mol Genet 14:1449-1455(2005).  
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