Transferrin, human ELISA Core Kit 10 plates K0231114

Cat. No.	K0231114
Product	Human Transferrin ELISA Core Kit
Type	ELISA Kit
Quantity	1000well assay
Specificity	This Kit is used to quantitatively measure levels of Human Transferrin in serum or other biological samples.
Description	Human Transferrin ELISA Core Kit contains 4 core components required for the quantitative measurement of Human Transferrin in a sandwich ELISA format. All reagents are sufficient to assay Human Transferrin in approximately 1000 ELISA plate wells.
Kit Components	Goat anti-Human Transferrin, affinity purified (K0211828) Goat anti-Human Transferrin, HRP conjugate (K0211833) Human Transferrin Reference Serum (Standard) TMB Substrate Kit (K0331060)
Cross Reactivity	Human
Storage & Shelf life	1 year at 2-8°C
Buffer Preparation	1. Coating Buffer : 50 mM Carbonate-Bicarbonate Buffer, pH 9.6. Resolve the coating material (antigen or antibody) in the coating buffer to make 1ug/ml (1-10ug/ml). 2. Sample/Standard/Antibody Diluting Solution : Dilute Sample/Standard/Antibody in PBS (Phosphate Buffered Saline, pH7.4). Or use PBST (Washing solution) or Blocking solution instead of PBS to help prevent non-specific binding. 3. Washing Solution: Add 50% 1ml Tween 20 to 1 Liter PBS and mix well. 4. Color Reaction Mixture: Mix 1 volume of TMB Solution and 2 volume of Substrate Solution (H2O2) prior to use.
Sandwich ELISA Protocol	1. Coating 1) Dilute capture antibody (K0211828) at a ratio of 1:100 with Coating Buffer and for coat each well with 100 ul of diluted capture antibody. 2) Incubate for at least 1 hr at Room Temperature. (or 1-3 hours at room temperature / overnight at 4°C). 3) After incubation, Remove the solution of each well and fill up the Washing Solution. Repeat 3-5 times. 2. Blocking 1) Add 200 ul of Blocking Solution to each well. 2) Incubate for at least 1 hr at room temperature. 3) After incubation, Remove the solution of each well and fill up the Washing Solution. Repeat 3-5 times. 3. Reacting Standards and Samples 1) Dilute the standards and samples in Dilution Solution at 1:2 serial dilutions (ex : 250-3.9 ng/ml). 2) Transfer 100 ul of standard or sample to assigned wells. 3) Incubate for at least 1 hr at room temperature. 4) After incubation, remove samples and standards and wash each well 3-5 times. 4. Detection Antibody 1) Dilute the Detection Antibody (K0211833) in Antibody Dilution Solution. Working dilution range is 1:10,000 to 1:200,000. (Recommended starting dilution is 1:100,000.) 2) Add 100 ul per well and incubate for at least 1 hr at room temperature. 3) After incubation, Remove the solution of each well and fill up the Washing Solution. Repeat 3-5 times. 5. Color Reaction and Reading 1) Apply 150ul Color Reaction Mixture (50ul TMB solution and 100 ul Substrate Solution) to each well. Mix well by vortexing or shaking slightly. 2) After sufficient color development (5-10 minutes at room temperature or at 37°C), add 50ul Stop Solution (2M H2SO4) to each well. 3) Using a microtiter plate reader, read the plate at the wavelength that is appropriate for the substrate used (450 nm for TMB) 6. Calculation of Results 1) Average the duplicate readings from each standard, control, and sample. 2) Subtract the zero reading from each averaged value above. 3) Create a standard curve by reducing the data using ELISA reader's computer software capable of generating Standard curve-fit.