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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (AB133615)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (AB133615)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (AB133615)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (AB133615)

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 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (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.

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

CANX knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human CANX (Calnexin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-canx-calnexin-knockout-hek-293t-cell-line-ab255368'>ab255368</a>)

Predicted band size: 68 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (AB133615)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/5000 dilution

All lanes:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 68 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (AB133615)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/1000 dilution

All lanes:

HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 68 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (AB133615)

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.

All lanes:

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

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

Calnexin knockout HAP1 cell lysate at 20 µg

Lane 3:

THP1 cell lysate at 20 µg

Lane 4:

Raw264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate at 20 µg

Predicted band size: 68 kDa

false

• WB

Unknown

Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (AB133615)

All lanes:

Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/1000 dilution

Lane 1:

HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate at 10 µg

Lane 2:

A431 (Human epidermoid carcinoma cell line) cell lysate at 10 µg

Lane 3:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) cell lysate at 10 µg

Lane 4:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 68 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (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.

All lanes:

Western blot - Anti-Calnexin antibody [EPR3633(2)] - ER Membrane Marker (ab133615) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

Calnexin knockout HAP1 cell lysate at 20 µg

Lane 3:

THP1 cell lysate at 20 µg

Lane 4:

Raw264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate at 20 µg

Predicted band size: 68 kDa

false