All lanes : Anti-Macrophage Inflammatory Protein 1 alpha / CCL3 antibody [EPR16618-90] (ab179638) at 1/1000 dilution

Lane 1 : Untreated RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : RAW 264.7 treated with 100 ng/ml LPS for 3 hours and then 300 ng/ml Brefeldin A was added for the last 3 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

Developed using the ECL technique.

Predicted band size: 10 kDa
Observed band size: 10 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular mass observed is consistent with what has been described in the literature (PMID: 9848081).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) untreated or treated with 100 ng/ml Lipopolysaccharide for 3 hours, followed by 300 ng/ml Brefeldin A for 3 hours cells labeling Macrophage Inflammatory Protein 1 alpha / CCL3 with ab179638 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing positive staining in RAW 264.7 treated with 100 ng/ml Lipopolysaccharide for 3 hours, followed by 300 ng/ml Brefeldin A for 3 hours.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

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

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized RAW 264.7 (mouse  macrophage cell line transformed with Abelson murine leukemia virus) cell line, treated with 100 ng/ml Lipopolysaccharide for 3 hours, then add 300 ng/ml Brefeldin A for the last 3 hours (red) / Untreated control (green), labeling Macrophage Inflammatory Protein 1 alpha / CCL3 with ab179638 at 1/600 compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Macrophage Inflammatory Protein 1 alpha / CCL3 was immunoprecipitated from 0.35 mg RAW 264.7 (mouse  macrophage cell line transformed with Abelson murine leukemia virus), treated with 100 ng/ml Lipopolysaccharide for 3 hours, then add 300 ng/ml Brefeldin A for the last 3 hours, whole cell lysate with ab179638 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab179638 at 1/1000 dilution. VeriBlot for IP Detection Reagent(HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: RAW 264.7 treated with 100 ng/ml Lipopolysaccharide for 3 hours, then add 300 ng/ml Brefeldin A for the last 3 hours whole cell lysate 10 µg (Input).

Lane 2: ab179638 IP in RAW 264.7 treated with 100 ng/ml Lipopolysaccharide for 3 hours, then add 300 ng/ml Brefeldin A for the last 3 hours whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab179638 in RAW 264.7 treated with 100 ng/ml Lipopolysaccharide for 3 hours, then add 300 ng/ml Brefeldin A for the last 3 hours whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 10 seconds.

High sensitivity ECL substrate used to develop the blot.

Standard Curve for Macrophage Inflammatory Protein 1 alpha / CCL3 (Analyte: Recombinant mouse Macrophage Inflammatory Protein 1 alpha / CCL3 protein) dilution range 0-5 ng/mL using Capture antibody at 0.2 ug/mL and Detector Antibody at 0.5 ug/mL. Secondary antibody: Peroxidase Streptavidin SA-HRP at 1/20000 dilution. Concentration of ab179638 may vary from lot to lot; please use this curve as guideline.

Washing buffer: 1X PBST
Diluting/Blocking buffer and concentration: 1% BSA/PBS