Recombinant Anti-CCL4/MIP-1 beta antibody [EP521Y] (ab45690)

False colour image of Western blot: Anti-CCL4/MIP-1 beta antibody [EP521Y] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab45690 was shown to bind specifically to CCL4/MIP-1 beta. A band was observed at 12 kDa in wild-type THP-1 cell lysates with no signal observed at this size in CCL4 knockout cell line ab273719 (knockout cell lysate ab275512). To generate this image, wild-type and CCL4 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Blocking/Diluting buffer and concentration 5% NFDM /TBST 

Immunocytochemistry/Immunofluorescence analysis of THP-1 (Human monocytic leukemia cell line) cells labeling CCL4/MIP-1 beta + CCL4L with ab45690 at 1/100. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counter-stained with ab7291, ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution. DAPI was used to stain nuclei blue.

The expression increased after treatment with Lipopolysaccharides (LPS), 100 ng/mL for 4 hours, followed by addition of Brefeldin A (1 μg/mL) for 3 hours.

Blocking/Diluting buffer and concentration 5% NFDM /TBST.

CCL4/MIP-1 beta is induced in macrophages following exposure to bacterial LPS (PMID: 9848081).

ab45690 at 1/60 immunoprecipitating CCL4/MIP-1 beta + CCL4L in THP-1 (Human monocytic leukemia cell line) whole cell lysate observed at 12 KDa (lanes 1 and 2).

Lane 1 (input): THP-1 treated with 100 nM PMA overnight, then treated with 100 ng/mL LPS for 7 hours and 1 μg/mL Brefeldin A was added for the last 3 hours whole cell lysate, 10μg

Lane 2 (+): ab45690 + THP-1 treated with 100 nM PMA overnight, then treated with 100 ng/mL LPS for 7 hours and 1 μg/mL Brefeldin A was added for the last 3 hours whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab45690 in THP-1 treated with 100 nM PMA overnight, then treated with 100 ng/mL LPS for 7 hours and 1 μg/mL Brefeldin A was added for the last 3 hours whole cell lysate

For western blotting, ab45690 at 1/1000 and ab131366 VeriBlot for IP Detection Reagent (HRP) was used for detection (1/1000).

Blocking/Diluting buffer 5% NFDM/TBST

Blocking/Diluting buffer and concentration 5% NFDM /TBST