Recombinant Anti-MIP-1 alpha/CCL3 + CCL4/MIP-1 beta + CCL3L1 + CCL4L1 antibody [EPR19900-275] (ab206429)

All lanes : Anti-MIP-1 alpha/CCL3 + CCL4/MIP-1 beta + CCL3L1 + CCL4L1 antibody [EPR19900-275] (ab206429) at 1/1000 dilution

Lane 1 : Untreated THP-1 (human monocytic leukemia cell line) whole cell lysate
Lane 2 : THP-1 (1 treated with 80nM TPA overnight, then 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 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 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.

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 ab206429 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 cells treated with TPA (80nM overnight), then treated with 100ng/ml LPS for 3h and together with 300ng/ml BFA for another 3h. The nuclear counterstain 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.

All lanes : Anti-MIP-1 alpha/CCL3 + CCL4/MIP-1 beta + CCL3L1 + CCL4L1 antibody [EPR19900-275] (ab206429) 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

Predicted band size: 10 kDa

Exposure time:
Lanes 1-7 (left image): 180 seconds
Lane 6 (right image): 5 seconds

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 0.1% Tween 20 permeabilized THP-1 (human monocytic leukemia cell line) (treated with 80nM TPA overnight, then treated with 100ng/ml LPS for 3h and together with 300ng/ml BFA for another 3 hr) (red) or untreated (green) cells labeling Macrophage Inflammatory Protein 1 alpha / CCL3 with ab206429 at 1/600 dilution compared with a 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 H&L (Alexa Fluor® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.