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

7 product images

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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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 12 kDa

• 

  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) (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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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

• 

  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

• 

  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) (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) (Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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) (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.

• 

  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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 12 kDa

  Exposure time: 3min

• 

  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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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 (Goat Anti-Rabbit IgG Fc (Alexa Fluor® 488) preadsorbed ab150097), at 1/5000 dilution was used as the secondary antibody.
  

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  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 (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 (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.