Anti IgG (H&L)

Properties of DyLightTM Conjugates.

Rockland Rabbit anti-Transferrin (109-4134, lot 3033, green), Goat Anti-Alpha-1-Anti-Trypsin (100-101-147, lot 5842), and Mouse Anti-GST (200-301-200, lot 24882) were used in a multiplex system to detect target proteins under reducing (R) conditions (+4% BME) in albumin depleted human serum with 320ng of added GST. Sample was run by SDS-PAGE, transferred to 0.2 um PVDF using the BioRad Trans-Blot Turbo and blocked in 2.5% Blotto, 2.5% BSA, 0.02% Tween overnight at 4C. Membrane was probed with three primary antibodies at 1:1000 dilution (in MB-070 overnight at 4C). Detection shown was using DyLight549 Donkey anti-Rabbit IgG (611-742-127, lot 21100, shown as green), DyLight 488 Donkey anti-Mouse IgG (610-741-124, lot 21095, shown as blue), and DyLight 649 Donkey anti-Goat IgG (605-743-125, lot 20834, shown as red) at 1:10000 (in MB-070 30 min RT). Blots were washed, rinsed in methanol, dried and Images were collected using the BioRad VersaDoc System.

DyLight？ dyes can be used for two-color Western Blot detection with low background and high signal.？ Anti-tubulin was detected using a DyLight？ 549 conjugate.？ Anti-TNFa was detected using a DyLight？ 649 conjugate. The image was captured using the Typhoon？ 9410 Imaging System.

Western Blot of Anti-Rabbit IgG (H&L) (DONKEY) Antibody (Min X Bv Ch Gt GP Ham Hs Hu Ms Rt & Sh Serum Proteins) (p/n 611-701-127). Lane M: 3 μl Molecular Ladder. Lane 1: Rabbit IgG whole molecule (p/n 011-0102). Lane 2: Rabbit IgG F(ab) Fragment (p/n 011-0105). Lane 3: Rabbit IgG F(c) Fragment (p/n 010-0103). Lane 4: Rabbit IgM Whole Molecule (p/n 011-0107). Lane 5: Normal Rabbit Serum (p/n B309). All samples were reduced. Load: 50 ng of IgG, F(ab), F(c) and Serum, 25 ng of IgM. Block: MB-070 for 30 min at RT. Primary Antibody: Anti-Rabbit IgG (H&L) (DONKEY) Antibody (Min X Bv Ch Gt GP Ham Hs Hu Ms Rt & Sh Serum Proteins) (p/n 611-701-127) 1:7,500 for 60 min at RT. Secondary antibody: Anti-Donkey IgG (GOAT) Peroxidase Conjugated Antibody (p/n 616-1302) 1:40,000 in MB-070 for 30 min at RT. Predicted/Observed Size: 25 and 50 kDa for Rabbit IgG and Serum, 25 kDa for F(c) and F(ab), 70 and 23 kDa for IgM. Rabbit F(c) migrates slightly higher.

Properties of DyLight？ Conjugates.