Alexa Fluor® 647 Anti-Calreticulin antibody [EPR3924] - ER Marker

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Alexa Fluor® 647 Anti-Calreticulin antibody [EPR3924] - ER Marker (AB196159)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-CALR knockout cells (red line) stained with ab196159. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab196159, 0.1μg/ml dilution) for 30 min at 22°C.

A rabbit monoclonal IgG isotype control antibody (ab199093) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-CALR knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5,000 events were collected using a 40 mW Red laser (640nm) and 670/14 bandpass filter.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Alexa Fluor® 647 Anti-Calreticulin antibody [EPR3924] - ER Marker (AB196159)

Overlay histogram showing HeLa cells stained with ab196159 (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 (ab196159, 1/50 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG [EPR25A] Alexa Fluor® 647 (ab199093) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a solid-state 25mW red diode laser (635 nm) and 675/30 bandpass filter.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 647 Anti-Calreticulin antibody [EPR3924] - ER Marker (AB196159)

ab196159 staining Calreticulin in wild-type HAP1 cells (top panel) and CALR 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 ab196159 at 1/500 dilution (shown in red) and ab195887 at 1/250 dilution (shown in green) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 647 Anti-Calreticulin antibody [EPR3924] - ER Marker (AB196159)

ab196159 staining Calreticulin in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabiliszd in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab196159 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 488, shown in green) at 2μg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

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