All lanes : Anti-p57 Kip2 antibody [EP2515Y] (ab75974) at 1/500 dilution (purified)

Lane 1 : Mouse brain
Lane 2 : Rat brain

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 32 kDa
Observed band size: 57 kDa why is the actual band size different from the predicted?

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling p57 Kip2 with purified ab75974 at 1/250. 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.

Control: primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human placenta tissue labelling p57 Kip2 with purified ab75974 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 p57 Kip2 (red) with ab75974 at a 1/350 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with primary and secondary antibodies.

ab75974 (purified) at 1/30 immunoprecipitating p57 Kip2 in HeLa cells treated with dexamethasone (Lane 1). Lane 2 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-p57 Kip2 antibody [EP2515Y] (ab75974) at 1/500 dilution (purified) + Human brain at 20 µg

Secondary
HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 32 kDa
Observed band size: 57 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-p57 Kip2 antibody [EP2515Y] (ab75974) at 1/500 dilution (unpurified)

Lane 1 : HeLa lysate
Lane 2 : Jurkat lysate
Lane 3 : Human brain lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 32 kDa
Observed band size: 57 kDa why is the actual band size different from the predicted?

ab75974 staining p57 Kip2 in HeLa cells treated with dexamethasone (ab120743), by ICC/IF. Increase in p57 Kip2 expression correlates with increased concentration of dexamethasone as described in literature.
The cells were incubated at 37°C for 24h in media containing different concentrations of ab120743 (dexamethasone) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with unpurified ab75974 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.