All lanes : Anti-CCL4/MIP-1 beta antibody [EP521Y] (ab45690) at 1/1000 dilution

Lane 1 : Untagged human CCL4 recombinant protein (aa24-92)
Lane 2 : Untagged human CCL4L recombinant protein (aa24-92)
Lane 3 : GST-tagged human CCL3 recombinant protein (aa27-92)
Lane 4 : GST-tagged human CCL3L recombinant protein 2*(aa28-93)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 10 kDa
Observed band size: 12 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

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.

All lanes : Anti-CCL4/MIP-1 beta antibody [EP521Y] (ab45690) at 1/1000 dilution

Lane 1 : Untreated THP-1 (human acute monocytic leukemia) lysate
Lane 2 : THP-1 treated with 100 nM Phorbol-12-myristate-13-acetate(PMA) overnight, then treated with Lipopolysaccharides (LPS) 100 ng/mL for 7 hours and then 1 µg/mL Brefeldin A was added for the last 3 hours, lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Predicted band size: 10 kDa
Observed band size: 12 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

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

All lanes : Anti-CCL4/MIP-1 beta antibody [EP521Y] (ab45690) at 1/1000 dilution

Lane 1 : Untreated Raw264.7 (mouse abelson murine leukemia virus-induced tumor) whole cell lysate
Lane 2 : Raw264.7 (mouse abelson murine leukemia virus-induced tumor) treated with LPS 10µg/mL for 4 hours and then 1 µg/mL Brefeldin A was added for the last 3 hours lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 10 kDa


Exposure time: 3 minutes

Blocking/Diluting buffer and concentration 5% NFDM /TBST 

ELISA analysis of Human CCL4/MIP-1 beta recombinant protein at 500 ng/mL with ab45690. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.