Benchmarking
Fluorescent Western blot with Cyanagen IR-BLOT and LI-COR IRDye® Secondary antibodies. Sample: 2-fold dilution series of HeLa whole cell lysate (abcam®) from 5μg to 0.078 μg of total protein. Membrane: Trans-Blot® Turbo™ Mini Nitrocellulose Transfer Packs (Bio-Rad). Blocking: 3% ECL™ Blocking Agent (GE Healthcare) in PBS. Primary antibody: Rabbit anti-Human HSP-90 (Santa Cruz Biotechnology) 1:4000 or Mouse anti-Human Vinculin (Sigma) 1:5000. Secondary antibody: A) IR-BLOT 800 Goat anti-Mouse (Cyanagen); B) IR-BLOT 800 Goat anti-Rabbit (Cyanagen); C) IR-BLOT 700 Goat anti-Mouse (Cyanagen); D) IR-BLOT 700 Goat anti-Rabbit (Cyanagen); E) IRDye® 800CW Goat anti-Mouse (LI-COR); F) IRDye® 800CW Goat anti-Rabbit (LI-COR); G) IRDye® 680RD Goat anti-Mouse (LI-COR); H) IRDye® 680RD Goat anti-Rabbit (LI-COR); 1:25000. Imager: ODISSEY ® CLX – LI-COR.

Increased Quantification Accuracy
Enhanced accuracy in Western blot using IR-BLOT Secondary Antibodies. A) Western blot detection of vinculin and HSP-90 on HeLa cell lysate with IR-BLOT Secondary antibodies on 2-fold dilutions of HeLa whole cell lysate. Results are the mean of six replicates. Blots were incubated with Rabbit-anti Human HSP-90 (1:4000) and Mouse anti-vinculin (1:5000). Imager: ODISSEY ® CLX – LI-COR. B) Integrated signal intensity. Data are the mean of six replicates. R2= coefficient of determination.

High sensitivity
Low background for high sensitive detection with IR-BLOT secondary antibodies. Western blot detection of HDAC-1 or vinculin on HeLa cell lysate with mid-level ECL substrates (WESTAR Antares – Cyanagen, Amersham ECL Prime – GE Healthcare) or IR-BLOT secondary antibodies. Blots were incubated with Mouse anti-vinculin 1:4000 (A) or Rabbit-anti Human HDAC-1 1:2500 (B) and Secondary antibody-HRP or IR-BLOT Secondary antibody 1: 100000. The signal-to-noise ratio (S/N) was calculated on at least three replicates.

Extreme signal duration
Signal duration of IR-BLOT over time. Six replicate of Western blots, containing 2-fold dilution series of HeLa whole cell lysate incubated with Mouse anti-Vinculin 1:5000 and IR-BLOT 800 Goat anti-Mouse 1:25000 and were simultaneously imaged with Odyssey Clx (LI-COR) immediately after the experiment (T0) and after one week with membranes stored at 4°C.