All lanes : Anti-Macrophage Inflammatory Protein 1 alpha / CCL3 antibody [EPR22529-19] (ab229900) at 1/1000 dilution

Lane 1 : Untreated THP-1 (human monocytic leukemia cell line) whole cell lysate
Lane 2 : THP-1 (treated with 100 ng/ml phorbol-12-myristate-13-acetate (PMA) for 56 hours, followed by adding 1 µg/ml lipopolysaccharide (LPS) for a further 16 hours ) whole cell lysate

Lysates/proteins at 20 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: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

This blot was developed using a higher sensitivity ECL substrate.

The molecular weight observed is consistent with what has been described in the literature (PMID: 11369791)

Macrophage Inflammatory Protein 1 alpha / CCL3 was immunoprecipitated from 0.35 mg of THP-1 (human monocytic leukemia cell line) (treated with Phorbol-12-myristate-13-acetate (100ng/ml) for 56h, followed by adding Lipopolysaccharide (1ug/ml) for a further 16h ) whole cell lysate with ab229900 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab229900 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: THP-1 (human monocytic leukemia cell line) (treated with Phorbol-12-myristate-13-acetate (100ng/ml) for 56h, followed by adding Lipopolysaccharide (1ug/ml) for a further 16h ) whole cell lysate 10 μg (Input).
Lane 2: ab229900 IP in THP-1 (treated as above) whole lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab229900 in THP-1 (treated as above) whole cell lysate.

Blocking and dilution buffer and concentration: NFDM/TBST.
Exposure time: 40 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized THP-1 (human monocytic leukemia cell line) (untreated, green) / (treated with Phorbol-12-myristate-13-acetate (100ng/ml) for 56h, followed by adding Lipopolysaccharide (1ug/ml) for a further 16h, red) cells labeling Macrophage Inflammatory Protein 1 alpha / CCL3 with ab229900 at 1/500 dilution compared with a Rabbit monoclonal IgG Isotype control details (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG Fc (Alexa Fluor^® 488) preadsorbed (ab150097), at 1/5000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized THP-1 (human monocytic leukemia cell line) cells labeling Macrophage Inflammatory Protein 1 alpha / CCL3 with ab229900 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in THP-1 treated with Phorbol-12-myristate-13-acetate (100ng/ml) for 56 h, followed by adding Lipopolysaccharide (1ug/ml) for a further 16 h. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Treated: Cells treated with Phorbol-12-myristate-13-acetate (100ng/ml) for 56 h, followed by adding Lipopolysaccharide
(1ug/ml) for a further 16 h.