重组 Anti-CCL3 / MIP-1 alpha 抗体[EPR16618-90]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CCL3 / MIP-1 alpha antibody [EPR16618-90] (AB179638)

Intracellular 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CCL3 / MIP-1 alpha antibody [EPR16618-90] (AB179638)

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 Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CCL3 / MIP-1 alpha antibody [EPR16618-90] (AB179638)

Flow cytometry staining of C57BL/6 Mouse Bone Marrow Cell-derived Macrophages (top) or C57BL/6 Mouse Bone Marrow Cell-derived Macrophages treated with LPS, 18 Hours, 10ng/mL + Brefeldin A, 3 Hours, 1μg/mL (bottom) with ab179638 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). Cells were fixed and permeabilised with BD Cytofix/Cytoperm™ for 20 min. Cells were incubated for 30min on ice in 1x PBS containing 10μg/ml anti CD16/CD32 and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab179638 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶in 100 μl at 5.0 μg/ml (1/100)) for 30min at 22°C. The cells were simultaneously stained with F4/80.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• IP

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Immunoprecipitation - Anti-CCL3 / MIP-1 alpha antibody [EPR16618-90] (AB179638)

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.

All lanes:

Immunoprecipitation - Anti-CCL3 / MIP-1 alpha antibody [EPR16618-90] (ab179638)

Predicted band size: 10 kDa

false

• sELISA

Lab

Sandwich ELISA - Anti-CCL3 / MIP-1 alpha antibody [EPR16618-90] (AB179638)

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

• WB

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Western blot - Anti-CCL3 / MIP-1 alpha antibody [EPR16618-90] (AB179638)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-CCL3 / MIP-1 alpha 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 at 20 µg

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 at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 10 kDa,39 kDa

Observed band size: 10 kDa

true

Exposure time: 3min