Anti-CCL4/MIP-1 beta 抗体 [EP521Y] - BSA and Azide free (ab187674)

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

Lane 1 : Wild-type THP-1 Vehicle control + Brefeldin A (5 u/mL, 6 h) cell lysate
Lane 2 : Wild-type THP-1 Treated PMA (100 ng/mL, 56 h) + LPS (1 u/mL, 24 h) + Brefeldin A (5 u/mL, 6 h) cell lysate
Lane 3 : CCL4 knockout THP-1 Vehicle control + Brefeldin A (5 u/mL, 6 h) cell lysate
Lane 4 : CCL4 knockout THP-1 Treated PMA (100 ng/mL, 56 h) + LPS (1 u/mL, 24 h) + Brefeldin A (5 u/mL, 6 h) cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

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

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.

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

This data was developed using ab45690, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

This data was developed using ab45690, the same antibody clone in a different buffer formulation.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) cell 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

This data was developed using ab45690, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

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

This data was developed using ab45690, the same antibody clone in a different buffer formulation.

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 and dilution 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

This data was developed using ab45690, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

This data was developed using ab45690, the same antibody clone in a different buffer formulation.

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.