Anti-Calreticulin 抗体 [EPR3924] - ER Marker

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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 ab92516 for 30 mins at room temperature. 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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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.

• IHC

Lab

Immunohistochemistry - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

Immunohistochemical analysis of formalin fixed paraffin embedded human kidney labelling Calreticulin with ab92516 at a concentration of 0.004 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH 8.5 for 32 mins.

ab92516 anti-Calreticulin [EPR3924] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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).

Alexa Fluor^® 488 (ab196158) and Alexa Fluor^® 647 (ab196159) conjugated versions are available for this clone.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

ab92516 showing negative staining in Normal human heart tissue.

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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 Fluorr^® 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.

Alexa Fluorr^®488 (ab196158) and Alexa Fluorr^®647 (ab196159) conjugated versions are available for this clone.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

Overlay histogram showing HeLa cells stained with ab92516 (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 (ab92516, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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 ab92516 for 30 mins at room temperature. 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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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 ab92516 for 30 mins at room temperature. 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).

• WB

Supplier Data

Western blot - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

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

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

All lanes:

Western blot - Anti-Calreticulin antibody [EPR3924] - ER Marker (ab92516) at 1/10000 dilution

Lane 1:

HepG2 (Human hepatocellular carcinoma epithelial cell) Whole cell lysates at 15 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Lane 3:

Mouse brain lysates at 15 µg

Lane 4:

Rat brain lysates at 15 µg

Lane 5:

COS-1 (African green monkey kidney fibroblast-like) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 48 kDa

Observed band size: 55 kDa

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• WB

Lab

Western blot - 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.

All lanes:

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

Predicted band size: 48 kDa

Observed band size: 55 kDa

false

• WB

Unknown

Western blot - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

All lanes:

Western blot - Anti-Calreticulin antibody [EPR3924] - ER Marker (ab92516) at 1/1000 dilution

Lane 1:

SH-SY5Y cell lysate at 10 µg

Lane 2:

HL-60 cell lysate at 10 µg

Lane 3:

HepG2 cell lysate at 10 µg

Lane 4:

HeLa cell lysate at 10 µg

Lane 5:

Human fetal kidney lysate at 10 µg

Lane 6:

Human fetal brain lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 48 kDa

false

• WB

CiteAb

Western blot - Anti-Calreticulin antibody [EPR3924] - ER Marker (AB92516)

Calreticulin western blot using anti-Calreticulin antibody [EPR3924] - ER Marker ab92516. Publication image and figure legend from Ye, D., Zhu, J., et al., 2020, J Cancer, PubMed 31956372.

ab92516 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab92516 please see the product overview.

Silencing of CRT expression inhibits EMT, migration and invasion of NPC CNE2 cells. CNE2 cells were transfected with CRT-specific si-RNA (si-CRT) and si-Control, respectively. (A) Morphologies of si-CRT and si-Control NPC CNE2 cells. (B) The effect of Silencing of CRT expression on E-cadherin, vimentin, MMP-9 and TGF-β protein expression was measured by Western blot. (C) NPC CNE2 cell migration and invasion images and data analysis after Silencing of CRT (expression1x200). Data are shown as mean±SD. *p<0.05, **p<0.01, ***p<0.001.

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