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Dot Blot showing the detection of Rat IgG. A three fold serial dilution of Rat IgG starting at 200ng was spotted onto 0.45 ?m nitrocellulose. After blocking in Blocking Buffer for Fluorescent Western Blotting (p/n MB 070) 1 Hour at 20C, Anti Rat IgG (H&L) (RABBIT) Antibody DyLightTM 800 Conjugated (Min X Human Serum Proteins) (p/n 612 445 026) 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.
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-TNFa was detected using a DyLightTM 800 conjugate. The image was captured using the OdysseyR Infrared Imaging System developed by LI-COR.
Properties of DyLight？ Conjugates.
Western Blot of Anti-Rat IgG (H&L) (RABBIT) Antibody (Min X Human Serum Proteins) (p/n 612-4126). Lane M: 3 μl Molecular Ladder. Lane 1: Rat IgG whole molecule (p/n 012-0102). Lane 2: Rat IgG F(c) Fragment (p/n 012-0103). Lane 3: Rat IgG Fab Fragment (p/n 012-0105). Lane 4: Rat IgM Whole Molecule (p/n 012-0107). Lane 5: Rat Serum (p/n D310-05). All samples were reduced. Load: 50 ng per lane. Block: MB-070 for 30 min at RT. Primary Antibody: Anti-Rat IgG (H&L) (RABBIT) Antibody (Min X Human Serum Proteins) (p/n 612-4126) 1:1,000 for 60 min at RT. Secondary Antibody: Anti-Rabbit IgG (GOAT) Peroxidase Conjugated Antibody (p/n 611-103-122) 1:40,000 in MB-070 for 30 min at RT. Predicted/Observed Size: 25 and 55 kDa for Rat IgG and Serum, 25 kDa for F(c) and Fab, 78 and 25 kDa for IgM. Rat F(c) migrates slightly higher.
Dot Blot showing the detection of Rat IgG. A three-fold serial dilution of Rat IgG starting at 200ng was spotted onto 0.45 ?m nitrocellulose. After blocking in Blocking Buffer for Fluorescent Western Blotting (p/n MB-070) 1 Hour at 20°C, Anti-Rat IgG (H&L) (RABBIT) Antibody DyLight？ 800 Conjugated (Min X Human Serum Proteins) (p/n 612-445-026) 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.
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