All lanes : Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CANX knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 68 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (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.

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133615).

Immunohistochemical staining of paraffin embedded rat cardiac muscle 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133615).

All lanes : Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : Calnexin knockout HAP1 cell lysate
Lane 3 : THP1 cell lysate
Lane 4 : RAW 264.7 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 68 kDa

This WB data was generated using the same anti-Calnexin antibody clone [EPR3633(2)] in a different buffer formulation (cat# ab133615).

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.

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133615).

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) 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 Fluor^® 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133615).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133615).

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