Calreticulin Recombinant Rabbit Monoclonal Antibody [SU37-03]

Application: Immunocytochemistry (IF-cell)

Species: Human
Sample: HeLa (Human cervix adenocarcinoma epithelial cell)

Fixation: Ice cold 100% methanol, 5 minutes at room temperature.
Permeabilization: /

Blocking: 1% BSA + 10% normal goat serum, 1 hour at room temperature.
Antibody dilution buffer: 1% BSA in PBST.
Primary antibody: ET1608-60, 1/5,000, overnight at 4℃.
Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 45 minutes at room temperature.

Counterstain: Beta tubulin (HA601187, Red), 1/100, overnight at 4℃. The Nuclear counterstain was DAPI (Blue).

Western blot analysis of Calreticulin on different lysates with Rabbit anti-Calreticulin antibody (ET1608-60) at 1/50,000 dilution and competitor's antibody at 1/5,000 dilution.

Lane 1: HepG2 cell lysate
Lane 2: HeLa cell lysate
Lane 3: HL-60 cell lysate
Lane 4: C2C12 cell lysate
Lane 5: C6 cell lysate
Lane 6: Mouse liver tissue lysate
Lane 7: Rat liver tissue lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 48 kDa
Observed band size: 55 kDa

Exposure time: 1 minute 2 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1608-60) at 1/50,000 dilution and competitor's antibody at 1/5,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Knockdown (KD)

Western blot analysis of Calreticulin on different lysates with Rabbit anti-Calreticulin antibody (ET1608-60) at 1/50,000 dilution.

Lane 1: HAP1-parental cell lysate (10 µg/Lane)
Lane 2: HAP1-Calreticulin KD cell lysate (10 µg/Lane)

Predicted band size: 48 kDa
Observed band size: 55 kDa

Exposure time: 30 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1608-60) at 1/50,000 dilution was used in K1803 at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Knockdown (KD)

Western blot analysis of Calreticulin on different lysates with Rabbit anti-Calreticulin antibody (ET1608-60) at 1/50,000 dilution.

Lane 1: HeLa-si NT cell lysate (10 µg/Lane)
Lane 2: HeLa-si Calreticulin cell lysate (10 µg/Lane)

Predicted band size: 48 kDa
Observed band size: 55 kDa

Exposure time: 13 seconds; ECL: K1801;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1608-60) at 1/50,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-Calreticulin antibody (ET1608-60) at 1/5,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1608-60) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded human liver carcinoma tissue with Rabbit anti-Calreticulin antibody (ET1608-60) at 1/5,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1608-60) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Flow cytometric analysis of HeLa cells labeling Calreticulin.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1608-60, 1/2,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Calreticulin was immunoprecipitated in 0.2mg HeLa cell lysate with ET1608-60 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1608-60 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HeLa cell lysate (input)
Lane 2: Rabbit IgG instead of ET1608-60 in HeLa cell lysate
Lane 3: ET1608-60 IP in HeLa cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 24 seconds