Recombinant Anti-Calnexin antibody [EPR3632] (ab92573)

Lanes 1 - 4: Merged signal (red and green). Green - ab92573 observed at 80 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab92573 was shown to react with Calnexin in wild-type HEK-293T cells in Western blot with loss of signal observed in CANX knockout cell line ab255368 (CANX knockout cell lysate ab263805). Wild-type HEK-293T and CANX knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab92573 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 20000 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.

ab92573 staining Calnexin in wild-type HAP1 cells (top panel) and CANX knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 ab92573 at 1/1000 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) 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). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Lanes 1 - 4: Merged signal (red and green). Green - ab92573 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab92573 was shown to specifically react with Calnexin when Calnexin knockout samples were used. Wild-type and Calnexin knockout samples were subjected to SDS-PAGE. ab92573 and ab8245 (loading control to GAPDH) were diluted at 1/20,000 and 1/10,000 respectively 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 1/10,000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin embedded Human tonsil tissue using ab92573 at a 1/100 dilution.

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

Calnexin was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg with 92573 at 1/100 dilution (2µg). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg

Lane 2: ab92573 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab92573 in HeLa whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Lanes 1 - 2: Merged signal (red and green). Green - ab92573 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab92573 and a competitor's rabbit polyclonal antibody.