Anti-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19] (AB229900)

Intracellular flow cytometric analysis of4% 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19] (AB229900)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19] (AB229900)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) (top) or PBMCs treated with LPS 1μg/mL and Brefeldin 1μg/mL for 18 Hours (bottom), with ab229900 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left). Cells were fixed and permeabilised with BD Cytofix/Cytoperm™ for 20 min. PBMCs were incubated for 30 mins on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab229900 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (1x 10⁶ in 100 µl at 0.04 μg/ml (1/12500 dilution)) for 30 mins at 22°C . The cells were simultaneously stained with CD14.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 dilution for 30 mins 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-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19] (AB229900)

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.

All lanes:

Immunoprecipitation - Anti-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19] (ab229900)

Observed band size: 12 kDa

false

• WB

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Western blot - Anti-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19] (AB229900)

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)

All lanes:

Western blot - Anti-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19] (ab229900) at 1/1000 dilution

Lane 1:

Untreated THP-1 (human monocytic leukemia cell line) whole cell lysate at 20 µg

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 at 20 µg

Secondary

All lanes:

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

Observed band size: 12 kDa

false

Exposure time: 3min

• WB

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Western blot - Anti-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19] (AB229900)

Blocking / Dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19] (ab229900) 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='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 12 kDa

false

• WB

Lab

Western blot - Anti-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19] (AB229900)

Exposure time :
Lane 1-5 20 seconds ; Lane 6-7 3 seconds

All lanes:

Western blot - Anti-MIP-1 alpha/CCL3 + CCL3L1 antibody [EPR22529-19] (ab229900) at 1/1000 dilution

Lane 1:

His-tagged mouse CCL3 Recombinant Protein

Lane 2:

His-tagged human CCL3 Recombinant Protein

Lane 3:

His-tagged human CCL4 Recombinant Protein

Lanes 4 - 5:

His-tagged human CCL18 Recombinant Protein

Lane 6:

GST-tagged human CCL3L1 Recombinant Protein

Lane 7:

GST-tagged human CCL4L1 Recombinant Protein

false