Recombinant Anti-Rab4 antibody [EPR3043] - Early Endosome Marker (ab109009)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Rab4 with Purified ab109009 at 1:170 dilution (10 µg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Rab4 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Human fetal brain lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab109009 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab109009 was shown to specifically react with Rab4 when Rab4 knockout samples were used. Wild-type and Rab4 knockout samples were subjected to SDS-PAGE. ab109009 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW)  preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD)  preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

ab109009 (purified) at 1:80 dilution (2µg) immunoprecipitating Rab4 in MCF7 whole cell lysate.
Lane 1 (input): MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab109009 & MCF7 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109009 in MCF7 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Rab4 with Purified ab109009 at 1/200 dilution (1µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labelling Rab4 with purified ab109009 at 1/250. Cells were fixed with 100% methanol and permeabilized with 0.1% triton X-100. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.