All lanes : Anti-beta Synuclein antibody [EP1537Y] (ab76111) at 1/10000 dilution (purified)

Lane 1 : Human brain lysate
Lane 2 : Mouse brain lysate
Lane 3 : Rat brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human 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?

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebrum tissue sections labeling beta Synuclein with purified ab76111 at 1/50 dilution (2.4 µg/mL). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-beta Synuclein antibody [EP1537Y] (ab76111) at 1/10000 dilution (purified)

Lane 1 : His-Tagged human beta Synuclein (aa 1 to 134) recombinant protein
Lane 2 : His-Tagged alpha synuclein (aa 1 to 140) recombinant protein

Lysates/proteins at 0.015 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?

A: Anti-beta Synuclein antibody [EP1537Y] (ab76111)

B: Anti-Alpha-synuclein antibody [MJFR1] (ab138501) 

Immunocytochemistry/ Immunofluorescence analysis of PC-12 (Rat adrenal gland pheochromocytoma) cells labeling beta Synuclein with purified ab76111 at 1/50 dilution (2.4 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% TritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of PC-12 (Rat adrenal gland pheochromocytoma) cells labeling beta Synuclein with purified ab76111 at 1/100 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Purified ab76111 at 1/20 dilution (0.6 µg) immunoprecipitating beta Synuclein in Human brain lysate.
Lane 1 (input): Human brain lysate 10 µg
Lane 2 (+): ab76111 + Human brain lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76111 in Human brain lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 18 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebrum tissue sections labeling beta Synuclein with purified ab76111 at 1/50 dilution (2.4 µg/mL). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebrum tissue sections labeling beta Synuclein with purified ab76111 at 1/50 dilution (2.4 µg/mL). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.