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ELISA of Mouse anti 6xHIS Tag Antibody. Antigen: HIS tagged purified protein and E. coli cell lysates expressing HIS Tagged construct, GST and RON tagged purified proteins. Coating amount: 0.15ug per well. Primary antibody: 6xHIS Tag antibody at 100ug/mL. Dilution series: 2 fold. Mid point concentration: 200ng/mL. Secondary antibody: Peroxidase mouse secondary antibody at 1:10,000. Substrate: TMB (p/n TMBE 1000).
Properties of DyLightTM Conjugates.
DyLightTM dyes can be used for two-color Western Blot detection with low background and high signal. Anti-tubulin was detected using a DyLightTM 680 conjugate. Anti-TNFα was detected using a DyLightTM 800 conjugate. The image was captured using the OdysseyR Infrared Imaging System developed by LI-COR.
Western Blot showing detection of 6x HIS Epitope Tag. Lane 1: Molecular Weight Marker. Lane 2: Recombinant 6xHIS-SUMO-GFP. Lane 3: Nag1. Blocking with 5% Blotto (p/n B501-0500) 30 min at 20C. Detection: Anti-6x His Epitope Tag (MOUSE) Monoclonal Antibody DyLightTM 800 Conjugated (p/n 200-345-382) secondary antibody was used at 1:5000 in Blocking Buffer for Fluorescent Western Blotting (p/n MB-070) and imaged on the LiCor Odyssey imaging system.
Properties of DyLight? Conjugates.
Western Blot of Mouse anti-6xHIS Tag Antibody. Lane 1: 100ng Purified histidine-tagged recombinant protein. Lane 2: 200ng E. coli cell lysate containing histidine-tagged expression construct. Lane 3: 100ng Purified GST-tagged recombinant protein. Lane 4: 100ng Purified FLAG-tagged recombinant protein. Primary antibody: Mouse anti-6xHIS Tag antibody at 1:5,000 overnight at 4C. Secondary antibody: Peroxidase mouse secondary antibody at 1:20,000 for 30 min at RT. Block: 5% BLOTTO for 1 hr at RT.
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