Recombinant Anti-TGN46 antibody [EPR12676] (ab174280)

Lanes 1 - 4: Merged signal (red and green). Green - ab174280 observed at 80 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab174280 was shown to react with TGN46 in wild-type A549 cells in Western blot with loss of signal observed in TGOLN2 knockout cell line ab269510 (knockout cell lysate ab269672). Wild-type A549 and TGOLN2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab174280 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

Blocking buffer: 5% NFDM/TBST.

Exposure: 10 seconds, 100 seconds.

Immunoprecipitation. ab174280 at 1/1000 labeling TGN46 immunoprecipitated from Human fetal liver lysate using ab174280 at 1/10.

Immunohistochemical analysis of paraffin embedded Human brain tissue labeling TGN46 with ab174280 at 1/50.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded Human kidney tissue labeling TGN46 with ab174280 at 1/50.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab174280 staining TGN46 in HepG2 cells (top panel - positive control) and HeLa cells (bottom panel - negative control, low TGN46 expression). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab174280 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).