All lanes : Anti-Galectin 3 antibody [A3A12] (ab2785) at 1/1000 dilution

Lane 1 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate

Lysates/proteins at 25 µg/ml per lane.

Predicted band size: 26 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of human ovarian carcinoma tissue (right) labeling Galectin 3 in the nucleus and cytoplasm with ab2785, compared with a negative control (left) by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature. Tissue sections were incubated with the primary antibody (1:100 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-mouse IgG was used as the secondary antibody, followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Immunofluorescent analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Galectin 3 (green) in the cytoplasm and nucleus with ab2785. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Galectin 3 monoclonal antibody (Product # MA1-940) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 °C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x

Immunohistochemical analysis (formalin/PFA-fixed paraffin-embedded sections) human colon tissue (right) labelling Galectin 3 in the nucleus and cytoplasm with ab2785, compared with a negative control (left). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature. Tissue sections were incubated with the primary antibody (1:100 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-mouse IgG was used as the secondary antibody, followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Immunofluorescent analysis of NIH/3T3 (Mouse embryo fibroblast cell line) cells labelling Galectin 3 (green) in the cytoplasm and nucleus with ab2785. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with the primary antibody (1:100 in 3% BSA-PBS) overnight at 4 ºC. A DyLight-conjugated anti-mouse was used as the secondary antibody. Red (phalloidin) - F-actin, Blue - nuclei. Images were taken at a magnification of 60x.

Immunohistochemical analysis of (formalin/PFA-fixed paraffin-embedded sections) mouse colon tissue (right) labelling Galectin 3 in the nucleus and cytoplasm with ab2785, compared with a negative control (left) by . To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature. Tissue sections were incubated with the primary antibody (1:20 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-mouse IgG was used as the secondary antibody, followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Immunofluorescent analysis of MCF7 (Human breast adenocarcinoma cell line) cells labelling Galectin 3 (green) in the cytoplasm and nucleus with ab2785. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were incubated with the primary antibody (1:100 in 3% BSA-PBS) overnight at 4 ºC. A DyLight-conjugated anti-mouse was used as the secondary antibody. Red (phalloidin) - F-actin, Blue - nuclei. Images were taken at a magnification of 60x.

Immunofluorescent analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Galectin 3 with ab2785. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2785, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.