Immunocytochemistry/Immunofluorescence analysis of PC-12 cells labelling alpha + beta Synuclein with purified ab51252 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

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

Overlay histogram showing U87-MG cells fixed in 4% PFA and stained with purified ab51252 at a dilution of 1 in 30 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

All lanes : Anti-alpha + beta Synuclein antibody [EP1646Y] (ab51252) at 1/5000 dilution (purified)

Lane 1 : Mouse brain tissue lysate
Lane 2 : Rat brain tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 14 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM /TBST.

Anti-alpha + beta Synuclein antibody [EP1646Y] (ab51252) at 1/5000 dilution (purified) + Human cerebellum tissue lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 14 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM /TBST.

Flow Cytometry analysis of U87-MG cells labelling alpha + beta Synuclein with purified ab51252 at 1/30 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

Anti-alpha + beta Synuclein antibody [EP1646Y] (ab51252) at 1/50000 dilution (unpurified) + Human fetal brain tissue lysate at 10 µg

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

Predicted band size: 14 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Immunocytochemistry/Immunofluorescence analysis of rat primary hippocampal neurons, staining alpha + beta Synuclein with unpurified ab51252. Cells were fixed, permeabilized, and blocked with 10% donkey serum at room temperature. Cells were incubated with primary antibody (1/1000) at 4°C for 24 hours. A Cy2-conjugated donkey anti-rabbit IgG was used as the secondary antibody.

Overlay histogram showing SH-SY5Y cells stained with unpurified ab51252 (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 (unpurified ab51252, 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.