Tips for Using Human CCL5 ELISA Kit
Posted in the Biotech Forum
#1 Feb 20, 2013
Human CCL5 ELISA development kit contains the key components required for the quantitative measurement of natural and/or recombinant CCL5 in a sandwich ELISA format within the range of 32-3,000 pg/ml. Using the ELISA protocol described below; kit provides sufficient reagents to assay CCL5 in approximately 1500 ELISA plate wells.
Standard/Sample: Dilute standard from 2,000 pg/ml to zero in diluent. Immediately add 100 μl of standard or sample to each well in duplicate and incubate at room temperature for at least 2 hours.
Detection: Aspirate and wash plate 4 times. Dilute a portion of detection antibody in diluent to a concentration of 50 ng/ml. Add 100 μl per well and incubate at room temperature for 2 hours.
Avidin-HRP Conjugate: Aspirate and wash plate 4 times. Dilute 3.0 μl of avidin-HRP conjugate 1:5,000 (this dilution factor may require some optimization) in diluent for a total volume of 15 ml. Add 100μl per well and incubate at room temperature for 30 minutes.
TMB Liquid Substrate: Add 100 μl of TMB HRPO Microwell Substrate Standard Kinetic One Component "Ready Use" to each well. Incubate at room temperature for 20-30 minutes and monitor color development with an ELISA plate reader at 450 nm with wavelength correction set at 540 nm or 570 nm. Avoid placing plates in direct light.
Stop Solution: The reaction may be stopped after 15-20 minutes by adding 100 μl of 2 M sulfuric acid (Sigma Cat.# 339741) to each well. All eye, hand, face, and clothing protection should be used when working with sulfuric acid.
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