All lanes : Anti-Galectin 3 antibody [EP2775Y] (ab76245) at 1/10000 dilution (purified)

Lane 1 : A375 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : A431 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 26 kDa
Observed band size: 31 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung squamous carcinoma tissue labelling Galectin 3 with unpurified ab76245.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of HT-29 cells labelling Galectin with purified ab76245 at 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/500) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue labelling Galectin 3 with purified ab76245 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Galectin 3 with purified ab76245 at 1/50 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

All lanes : Anti-Galectin 3 antibody [EP2775Y] (ab76245) at 1/10000 dilution (purified)

Lane 1 : RAW264.7 cell lysate
Lane 2 : NIH/3T3 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 26 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Galectin 3 antibody [EP2775Y] (ab76245) at 1/10000 dilution (unpurified)

Lane 1 : A375 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : SW480 cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 26 kDa
Observed band size: 31 kDa why is the actual band size different from the predicted?

ICC/IF image of unpurified ab76245 stained Panc-1 cells. The cells were 4% formaldehyde 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 ab76245 at 10µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor^® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Overlay histogram showing THP1 cells stained with unpurified ab76245 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% human serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76245, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Immunohistochemical analysis of formaldehyde fixed mouse lung tissue sections labelling Galectin 3 with ab76245 at a dilution of 1/6000. The secondary antibody used was biotin conjugated goat anti rabbit IgG at a dilution of 1/300. Antigen retrieval was heat mediated using citric acid.

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