Recombinant Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615)

Lanes 1- 2: Merged signal (red and green). Green - ab133615 observed at 90 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab133615 was shown to react with Calnexin in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255368 (knockout cell lysate ab263805) was used. Wild-type HEK-293T and CANX knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab133615 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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.

Lanes 1 - 4: Merged signal (red and green). Green - ab133615 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab133615 was shown to recognize Calnexin when Calnexin knockout samples were used, along with additional cross-reactive bands. Wild-type and Calnexin knockout samples were subjected to SDS-PAGE. ab133615 and ab8245 (loading control to GAPDH) were diluted 1/1000 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 h at room temperature before imaging.

Lanes 1 - 4: Merged signal (red and green). Green - ab133615 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab133615 and a competitor's top cited rabbit polyclonal antibody.

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded human pancreas with purified ab133615 at a working dilution of 1 in 4000. The secondary antibody used is ab97051, a HRP goat anti-rabbit (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Overlay histogram showing HeLa cells stained with unpurified ab133615 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab133615, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions. 

Overlay histogram showingHeLa cells fixed in 80%methanol and stained with purified ab133615 at a dilution of 1 in 360 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 150. Rabbit monoclonal IgG was used as an isotype control (black line) and cells without incubation with antibody were used as a negative control (blue line). 

Immunohistochemical analysis of paraffin-embedded human kidney tissue labelled with unpurified ab133615 at 1/100 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.