Recombinant Anti-Alpha-synuclein antibody [EPR20535] - BSA and Azide free (ab225866)

This data was developed using ab212184, the same antibody clone in a different buffer formulation

Blocking/Dilution buffer: 5% NFDM/TBST.

Human Alpha-synuclein recombinant protein contain aa1-140 with His-tag. Human Beta-synuclein recombinant protein contain aa1-134 with His-tag.

This data was developed using ab212184, the same antibody clone in a different buffer formulation

Blocking/Dilution buffer: 5% NFDM/TBST.

The observed molecular weight is consistent with the literature (PMID: 11739566).

This data was developed using ab212184, the same antibody clone in a different buffer formulation:

False colour image of Western blot: Anti-Alpha-synuclein antibody [EPR20535] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab212184 was shown to bind specifically to Alpha-synuclein. A band was observed at 16 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in SNCA knockout cell line ab282333 (knockout cell lysate ab283006). To generate this image, wild-type and SNCA knockout U-87 MG cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

This data was developed using ab212184, the same antibody clone in a different buffer formulation:

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: SNCA (alpha Synuclein) knockout HAP1 whole cell lysate (20 µg)
Lane 3: Human brain whole tissue lysate (20 µg)
Lane 4: SH-SY5Y whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab212184 observed at 14 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab212184 was shown to specifically react with SNCA (alpha Synuclein) in wild type cells as signal was lost in SNCA (alpha Synuclein) knockout cells. Wild-type and SNCA (alpha Synuclein) knockout samples were subjected to SDS-PAGE. ab212184 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDy^e® 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab212184).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab212184).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab212184).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab212184).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse hippocampus tissue labeling Alpha-synuclein with ab212184 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Cytoplasmic staining on mouse hippocampus (PMID: 22112368).

The nuclear counterstain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab212184).

Alpha-synuclein was immunoprecipitated from 0.35 mg of mouse brain lysate with ab212184 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab212184 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: Mouse brainlysate 10ug (Input).

Lane 2: ab212184 IP in mouse brain lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab212184 in mouse brain lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab212184).