Recombinant Anti-Calreticulin antibody [EPR3924] - ER Marker (ab92516)

Lane 1: Wild-type HAP1 cell lysate (20 µg)

Lane 2: Calreticulin knockout HAP1 cell lysate (20 µg)

Lane 3: HeLa cell lysate (20 µg)

Lane 4: NIH3T3 cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab92516 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab92516 was shown to specifically react with Calreticulin when Calreticulin knockout samples were used. Wild-type and Calreticulin knockout samples were subjected to SDS-PAGE. ab92516 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

ab92516 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 ab92516 at 1/500 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).

Immunohistochemical analysis of paraffin-embedded Rat lung tissue labeling Calreticulin  with ab92516, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on rat lung.The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Exposure time: Lane 1 to 3: 10 seconds; Lane 4 and 5: 130 seconds

Overlay histogram showing HAP1 wildtype (green line) and HAP1-CALR knockout cells (red line) stained with ab92516. 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 (ab92516, 1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) preadsorbed (ab150081) at 1/2000 dilution for 30 min at 22°C.

A rabbit IgG isotype control antibody  (ab172730) 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 50 mW Blue laser (488nm) and 530/30 bandpass filter.

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Calreticulin  with ab92516, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on mouse liver.The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling Calreticulin with ab92516, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining human breast carcinoma.The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Formalin-fixed, paraffin-embedded normal human colon tissue stained for Calreticulin using ab92516 at 1/250 dilution in immunohistochemical analysis.

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

Immunocytochemistry/Immunofluorescence analysis of HT-29 (human colorectal adenocarcinoma) labelling Calreticulin with purified ab92516 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

Control: PBS only

ab92516, at 1/250 dilution, staining Calreticulin in paraffin embedded Human kidney tissue by Immunohistochemistry.

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

ab92516 showing negative staining in Normal human heart tissue.

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

Formalin-fixed, paraffin-embedded normal human liver tissue stained for Calreticulin using ab92516 at 1/250 dilution in immunohistochemical analysis.

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

Formalin-fixed, paraffin-embedded normal human placenta tissue stained for Calreticulin using ab92516 at 1/250 dilution in immunohistochemical analysis.

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

Formalin-fixed, paraffin-embedded normal human stomach tissue stained for Calreticulin using ab92516 at 1/250 dilution in immunohistochemical analysis.

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

Formalin-fixed, paraffin-embedded Papillary carcinoma of human thyroid gland tissue.stained for Calreticulin using ab92516 at 1/250 dilution in immunohistochemical analysis.

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