Recombinant Anti-Apolipoprotein E antibody [EP1374Y] (ab52607)

False colour image of Western blot: Anti-Apolipoprotein E antibody [EP1374Y] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab52607 was shown to bind specifically to Apolipoprotein E. A band was observed at 34-37 kDa in wild-type HepG2 cell lysates with no signal observed at this size in APOE knockout cell line. To generate this image, wild-type and APOE knockout HepG2 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Lanes 1-3: Merged signal (red and green). Green - ab52607 observed at 36 kDa. Red - loading control ab7291 observed at 50 kDa. 

 ab52607 Anti-Apolipoprotein E antibody [EP1374Y] was shown to specifically react with Apolipoprotein E in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266546 (knockout cell lysate ab256838) was used.  Wild-type and Apolipoprotein E knockout samples were subjected to SDS-PAGE.  ab52607 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively.  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 in 20000 dilution for 1 hour at room temperature before imaging.

Intracellular Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling Apolipoprotein E (red) with purified ab52607 (Unpurified) at a 1/250 dilution (10ug/mL). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti rabbit IgG (Alexa Fluorr^®488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue). 

Purified ab52607 at 1/20 dilution (0.5µg) immunoprecipitating Apolipoprotein E in HepG2 whole cell lysate.
Lane 1 (input): HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab52607 + HepG2 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52607 in HepG2 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/5000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 36 kDa

Purified ab52607 at 1/20 dilution (0.5µg) immunoprecipitating Apolipoprotein E in Human serum.
Lane 1 (input): Human serum 10µg
Lane 2 (+): ab52607 + Human serum.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52607 in Human serum.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 36 kDa