All lanes : Anti-HMGA1 antibody [EPR7839] (ab129153) (purified)

Lane 1 : HepG2 cell lysate
Lane 2 : SK-OV-3 cell lysate
Lane 3 : Caco-2 cell lysate
Lane 4 : BxPC-3 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: 12 kDa
Observed band size: 17 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.

Immunocytochemistry/Immunofluorescence analysis of BxPC-3 cells labelling HMGA1 with unpurified ab129153 at 1/250 dilution.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human transitional cell carcinoma of bladder tissue labelling HMGA1 with purified ab129153 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with Hematoxylin.

Anti-HMGA1 antibody [EPR7839] (ab129153) at 1/10000 dilution (purified) + Mouse brain tissue lysate at 10 µg

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

Predicted band size: 12 kDa
Observed band size: 17 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.

Anti-HMGA1 antibody [EPR7839] (ab129153) at 1/50000 dilution (purified) + Rat kidney tissue lysate at 10 µg

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

Predicted band size: 12 kDa
Observed band size: 17 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-HMGA1 antibody [EPR7839] (ab129153) at 1/10000 dilution (unpurified)

Lane 1 : SK-OV-3 cell lysate
Lane 2 : Caco-2 cell lysate
Lane 3 : BxPC-3 cell lysate
Lane 4 : HepG2 cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 12 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue labelling HMGA1 with unpurified ab129153 at 1/250.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling HMGA1 with purified ab129153 at 1/500. 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 (1/500) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Flow cytometry analysis of HepG2 cells labelling HMGA1 with purified ab129153 at 1/60 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

Overlay histogram showing HepG2 cells stained with unpurified ab129153 (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 / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab129153, 1/870 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. This antibody gave a positive signal in HepG2 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.