HRP Anti-Calreticulin antibody [EPR3924] - ER Marker

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HRP Anti-Calreticulin antibody [EPR3924] - ER Marker (AB195511)

IHC image of Calreticulin staining in a section of formalin-fixed paraffin-embedded human normal kidney*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4°C with ab195511 at 1μg/ml. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• WB

Lab

Western blot - HRP Anti-Calreticulin antibody [EPR3924] - ER Marker (AB195511)

ab195511 was shown to recognise Calreticulin in wild-type HAP1 cells as signal was lost at the expected MW in CALR knockout cells. Additional cross -reactive bands were observed in the wild0type and knockout cells. Wile-type and CALR knockout samples were subjected to SDS-PAGE. ab195511 was incubated overnight at 4°C at 1/5000 dilution. Blots were developed with ECL technique.

All lanes:

Western blot - HRP Anti-Calreticulin antibody [EPR3924] - ER Marker (ab195511) at 1/5000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

CALR knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 48 kDa

Observed band size: 55 kDa

true

Exposure time: 90s

• WB

Lab

Western blot - HRP Anti-Calreticulin antibody [EPR3924] - ER Marker (AB195511)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab195511 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

All lanes:

Western blot - HRP Anti-Calreticulin antibody [EPR3924] - ER Marker (ab195511) at 1/5000 dilution

Lane 1:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

Western blot - HeLa whole cell lysate (<a href='/en-us/products/cell-lysates/hela-whole-cell-lysate-ab150035'>ab150035</a>) at 10 µg

Lane 3:

Brain (Human) Tissue Lysate - fetal normal tissue at 10 µg

Predicted band size: 48 kDa

Observed band size: 55 kDa

true

Exposure time: 8s