Anti-Hsp27 antibody [G3.1] (ab2790) + HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Observed band size: 27 kDa why is the actual band size different from the predicted?

Immunohistochemistry of paraffin embedded human breast carcinoma with ab2790 labeling Hsp27 at 0.5μg/ml.

Immunohistochemistry of paraffin embedded human prostate carcinoma with ab2790 labeling Hsp27 at 0.5μg/ml.

 

 

Immunocytochemistry/immunofluorescence analysis of paraformaldehyde fixed MCF7 (human breast adenocarcinoma cell line) cells labelling Hsp27 with ab2790 at 2 µg/mL. Goat Anti-Mouse IgG was used as the secondary antibody (green). Nuclear DNA labelled red.

Anti-Hsp27 antibody [G3.1] (ab2790) at 1/1000 dilution + Mouse whole brain tissue lysate. at 10 µg

Secondary
An HRP-conjugated Goat polyclonal. at 1/10000 dilution

Developed using the ECL technique.

Observed band size: 24 kDa why is the actual band size different from the predicted?


Exposure time: 5 minutes


Blocking Step: 5% Milk for 1 hour at 25°C.
Gel Running Conditions: Reduced, Denaturing Bis-tris 4-12%

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Overlay histogram showing HeLa cells (ab150035) stained with ab2790 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum (ab7481)  / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2790, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.