Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Hsp27 knockout  HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: MCF7 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab78806 observed at 27 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab78806 was shown to specifically react with Hsp27 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when Hsp27 knockout samples were used. Wild-type and HSPB1 knockout samples were subjected to SDS-PAGE.  Ab78806 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ICC/IF image of ab78806 stained HeLa cells. 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 (ab78806, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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. This antibody also gave a positive result in 4% PFA fixed (10 min) HepG2 and MCF7 cells at 1µg/ml, and in 100% methanol fixed (5 min) HeLa and MCF7 cells at 1µg/ml.

All lanes : Anti-Hsp27 antibody (ab78806) at 1 µg/ml

Lane 1 : Human skeletal muscle tissue lysate - total protein (ab29330)
Lane 2 : Human heart tissue lysate - total protein (ab29431)
Lane 3 : Human liver tissue lysate - total protein (ab29889)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 23 kDa
Observed band size: 27 kDa why is the actual band size different from the predicted?
Additional bands at: 30 kDa, 60 kDa. We are unsure as to the identity of these extra bands.



Hsp27 contains a number of potential phosphorylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

IHC image of Hsp27 staining in human cervical carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab78806, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.