All lanes : Anti-Calreticulin antibody - ER Marker (ab2907) at 1/1000 dilution

Lane 1 : HL-60 cell lysate
Lane 2 : LNCaP cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : MCF-7 cell lysate
Lane 5 : Mouse liver tissue lysate
Lane 6 : Rat liver tissue lysate

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP at 1/4000 dilution

Observed band size: 55 kDa why is the actual band size different from the predicted?

ab2907 used at a 1/1000 dilution staining Calreticulin in HeLa cells by Immunocytochemistry/ Immunofluorescence.
HeLa cells were transfected overnight with empty vector or plasmids encoding the indicated IFITM3 constructs. Immunofluorescence with a-HA antibodies allowed IFITM3 visualization, and a-calreticulin staining allowed visualization of the ER. TOPRO-3 was used to visualize nuclei. Scale bars indicate 10 µm. Ub? indicates mutation of Lys-24, Lys-83, Lys-88, and Lys-104 to alanine.

Immunocytochemistry/Immunofluorescence analysis of Calreticulin (green) in A431 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were incubated with ab2907 (1:75) for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody (1:250) for 30 minutes at room temperature. Actin was stained with DyLight 650 Phalloidin (1:120) and nuclei (blue) were stained with Hoechst (1ug/ml) for 30 minutes. Images were taken at 20X magnification.

All lanes : Anti-Calreticulin antibody - ER Marker (ab2907) at 1/1000 dilution

All lanes : Whole cell lysate prepared from mouse skeletal muscle

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : HRP-conjugated mouse polyclonal to rabbit Ig at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Exposure time: 3 seconds

See Abreview

Immunocytochemistry/Immunofluorescence analysis of Calreticulin (red) in U2OS cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were incubated with ab2907 (1:75) for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 633 goat anti-rabbit IgG secondary antibody (1:250) for 30 minutes at room temperature. Actin was stained with DyLight 488 Phalloidin (1:300) and nuclei (blue) were stained with Hoechst (1ug/ml) for 30 minutes. Images were taken at 20X magnification.

Immunocytochemsitry/Immunofluorescence analysis of Calreticulin (green) U2OS cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA in PBS 0.1% triton-X for 30 minutes at room temperature. Cells were incubated with ab2907 (1:50) for at least 1 hour at room temperature. Cells were washed with PBS and incubated with DyLight 488 goat-anti-rabbit IgG secondary antibody (1:250) for 30 minutes at room temperature. Actin filaments (red) were stained with DyLight 554-Phalloidin (1:300) in PBS and incubated for 30 minutes. Nuclei (blue) were stained with Hoechst 33342 dye (1µg/mL). Images were taken at 20X magnification.

Immunocytochemistry/Immunofluorescence analysis of Calreticulin (green) in A431 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were incubated with ab2907 (1:75) for at least 1 hour at room temperature, washed with PBS, and incubated with Dylight 488 goat anti-rabbit IgG secondary antibody (1:250) for 30 minutes at room temperature. Actin was stained with Dylight 350 Phalloidin (1:120) and nuclei (red) were stained with DRAQ5 (1ug/ml) for 30 minutes. Images were taken at 20X magnification.

Immunocytochemistry/Immunofluorescence analysis of Calreticulin (green) in A431 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were incubated with ab2907 (1:75) for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody (1:250) for 30 minutes at room temperature. Actin was stained with DyLight 550 Phalloidin (1:120) and nuclei (blue) were stained with Hoechst (1ug/ml) for 30 minutes. Images were taken at 20X magnification.

Immunocytochemistry/Immunofluorescence analysis of HMVEC cells labelling Calreticulin using ab2907.

Immunofluorescence analysis of HepG2 cells, staining Calreticulin with ab2907.

Cells were fixed with paraformaldehyde, permeabilized with 0.1% Saponin and blocked with 10% serum for 1 hour at 20°C. Samples were incubated with primary antibody (1/200 in PBS + 0.1% saponin) for 1 hour at 20°C. An AlexaFluor®647-conjugated donkey anti-rabbit polyclonal IgG (1/400) was used as the secondary antibody.

See Abreview