ET1608-60

Calreticulin Recombinant Rabbit Monoclonal Antibody [SU37-03]

cat.: ET1608-60

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IHC-P, FC, IP
Clonality:	Monoclonal
Clone number:	SU37-03

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 48 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within Human Calreticulin aa 40-89 / 417.
Positive control:	HepG2 cell lysate, HeLa cell lysate, HL-60 cell lysate, C2C12 cell lysate, C6 cell lysate, mouse liver tissue lysate, rat liver tissue lysate, Hela, NIH/3T3 cell lysate, NIH/3T3, human liver tissue, human liver carcinoma tissue, human kidney tissue, mouse brain tissue.
Subcellular location:	Endoplasmic reticulum lumen, Cytoplasm, Secreted, Cell surface, Sarcoplasmic reticulum lumen, nucleus.
Recommended Dilutions: WB IF-Cell IHC-P FC IP	1:5,000-1:50,000 1:50-1:2,000 1:50 1:2,000 Use at an assay dependent concentration.
Uniprot #:	SwissProt: P27797 Human \| P14211 Mouse \| P18418 Rat
Alternative names:	Autoantigen RO CALR CALR protein CALR_HUMAN Calregulin Calreticulin cC1qR CRP55 CRT CRTC Endoplasmic reticulum resident protein 60 Epididymis secretory sperm binding protein Li 99n ERp60 FLJ26680 grp60 HACBP HEL S 99n RO Sicca syndrome antigen A (autoantigen Ro; calreticulin) Sicca syndrome antigen A SSA

Images

Fig1: 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;

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.

Fig2: Immunocytochemistry analysis of HeLa cells labeling Calreticulin with Rabbit anti-Calreticulin antibody (ET1608-60) at 1/2,000 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Calreticulin antibody (ET1608-60) at 1/2,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Fig3: Western blot analysis of Calreticulin on different lysates with Rabbit anti-Calreticulin antibody (ET1608-60) at 1/5,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HepG2 cell lysate
Lane 3: HL-60 cell lysate
Lane 4: NIH/3T3 cell lysate
Lane 5: C2C12 cell lysate
Lane 6: Mouse liver tissue lysate
Lane 7: Rat liver tissue lysate

Lysates/proteins at 20 µg/Lane.

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

Exposure time: 20 seconds;

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 1/5,000 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.

Fig4: Immunocytochemistry analysis of NIH/3T3 cells labeling Calreticulin with Rabbit anti-Calreticulin antibody (ET1608-60) at 1/50 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-Calreticulin antibody (ET1608-60) at 1/50 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 647, HA1127) were used as the secondary antibody at 1/1,000 dilution.

	Fig5: Immunohistochemical analysis of paraffin-embedded human liver tissue using anti-Calreticulin antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1608-60, 1/50) for 30 minutes 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.
	Fig6: Immunohistochemical analysis of paraffin-embedded human liver carcinoma tissue using anti-Calreticulin antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1608-60, 1/50) for 30 minutes 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.
	Fig7: Immunohistochemical analysis of paraffin-embedded human kidney tissue using anti-Calreticulin antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1608-60, 1/50) for 30 minutes 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.
	Fig8: Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-Calreticulin antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1608-60, 1/50) for 30 minutes 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.

	Fig9: 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).
	Fig10: Calreticulin was immunoprecipitated in 0.2mg HeLa cell lysate with ET1608-60 at 2 µg/25 µl agarose. 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
	Fig11: 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 Lane 2: HeLa-si Calreticulin cell lysate Lysates/proteins at 10 µg/Lane. Predicted band size: 48 kDa Observed band size: 55 kDa Exposure time: 13 seconds; 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.

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.