Anti IgG (H&L)

Rockland ATTO 647N conjugated anti rabbit antibody was used to detect anti Beta Actin antibody (p/n 600 401 886 lot 26928). Hela (Lane 1) and NIH 3T3 (Lane 2) Whole cell lysates were run on a 4 20% gel, transferred to nitrocellulose under standard conditions, and incubated with anti beta actin at a dilution of 1:2000 (ON 4C). For secondary antibody detection, blot was incubated for 1 hr RT simultaneously with: 1. Rockland ATTO 647N conjugated anti rabbit antibody (p/n 611 156 122 lot 26426C, 1:10000 in MB 070, Shown on Left and 2. Rockland HRP conjugated anti rabbit IgG (611 1322 lot 19247, 1:10000 in MB 070, shown on right) Blot was dried, imaged at a wavelength of 700 nm on a LiCor Odyssey reader, rewetted in TBS and imaged after 2 min with a 30 sec exposure time using Rockland Femtomax 110 super sensitive Chemiluminescent substrate using the Biorad Versa Doc Imaging System.

ATTO R dyes can be used for multicolor immunofluorescent detection with low background and high signal. Examples shown are: A. Tubulin in PtK2- male Rat Kangaroo Kidney Epithelial Cells was detected using ATTO 532 labeled secondary antibody. B. Muscle alpha-actin was stained with a mouse primary antibody and ATTO 488 anti-mouse IgG (green) while Cytokeratin was stained with polyclonal rabbit anti-cytokeratin and ATTO 647N anti-rabbit IgG (red). C. HUVEC (Human umbilical vein endothelial cells were stained with anti- Vimentin-ATTO 532 (green), anti-E-Cadherin-ATTO 655 (red) and DAPI (blue). D. Rat colon sections were stained with Anti-Aquaporin 3-ATTO 594 antibody. Hoechst 33342 (blue) is used as counterstain. Images provided courtesy of Dr. Jorg Reichwein, ATTO-TEC GmbH

Western Blot of Unconjugated Anti-Rabbit IgG (H&L) (GOAT) Antibody (Min X Bv, Ch, Gt, GP, Ham, Hs, Hu, Ms, Rt & Sh Serum Proteins) (p/n 611-101-122). 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 per lane. Block: MB-070 for 30 min at RT. Primary Antibody: Anti-Rabbit IgG (H&L) (GOAT) Antibody (Min X Bv, Ch, Gt, GP, Ham, Hs, Hu, Ms, Rt & Sh Serum Proteins) (p/n 611-101-122) 1:1,000 for 60 min at RT. Secondary antibody: Anti-Goat IgG (DONKEY) Peroxidase Conjugated Antibody (p/n CUST10) 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.