All lanes : Anti-Alpha-synuclein (phospho S129) antibody [EP1536Y] (ab51253) at 1/1000 dilution

Lane 1 : Rat brain lysates, the membrane was incubated with alkaline phosphatase.
Lane 2 : Rat brain lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/2000 dilution

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

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

Exposure time: 20 seconds

IHC image of alpha Synuclein (phospho S129) staining Human Parkinson Substantia Nigra tissue section*, previously antigen was retrieved by heat mediated with citrate buffer pH 6, fixed in formalin and embedded in paraffin. This section was incubated with ab51253 at 10 µg/mL for 15 mins at room temperature and detected using an HRP conjugated compact polymer system, performed on a Leica Bond™ system using the standard protocol F. DAB was used as the chromogen. Counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay performed on Human normal Substantia Nigra.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

All lanes : Anti-Alpha-synuclein (phospho S129) antibody [EP1536Y] (ab51253) at 1/1000 dilution

Lane 1 : Mouse brain lysates, the membrane was incubated with alkaline phosphatase
Lane 2 : Mouse brain lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/2000 dilution

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

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

Exposure time: 40 seconds

Expression of WT aSyn in the SN is toxic over time. Mouse brain sections immunostained for TH (red panels) and aSyn (using ab51253) (green panels) 1, 2 and 3 weeks after injection with vectors encoding for EGFP or WT aSyn. Scale bar for isolated channels 200 μm and for merged channels 100 μm. 

All lanes : Anti-Alpha-synuclein (phospho S129) antibody [EP1536Y] (ab51253) at 1/1000 dilution

Lane 1 : Sarkosyl-insoluble brain extract from mice transgenic for PrPA53T alpha-synuclein (line M83)
Lane 2 : Recombinant alpha-synuclein phosphorylated at S129, 3ng

Predicted band size: 14 kDa

Direct ELISA antibody dose-response curve using ab51253.

Antibody concentration of 0-5000 ng/mL.

Antigen (Alpha-synuclein (pS129) phospho peptide and Alpha-synuclein non-phospho peptide) concentration of 1000 ng/mL.

An alkaline phosphatase conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.

Dot blot analysis of human alpha Synuclein (pS129) peptide (Lane 1), huamn alpha Synuclein (unmodified) peptide (Lane 2) labelling alpha Synuclein (pS129) with ab51253 at a dilution of 1/1000. Peroxidase conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody at a dilution of 1/2500.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

α-Synuclein (α-syn) and S129-phosphorylated α-synuclein protein levels in SNCA/SNCA mouse brains after 12 days of treatment with 4mM ambroxol (Amb). (A) Western blotting for α-synuclein (using ab1903) and serine 129 (S129)-phosphorylated α-synuclein protein (using ab51253) in the brainstem (example blots shown).

IHC image of alpha Synuclein (phospho S129) staining in free floating tgM83^+/- or tgM83^+/+ mouse brain tissue. The section was incubated with ab51253, 1/5000, for 15 hours at 4^oC and detected using a Biotinylated conjugated Anti-Rabbit monocloal antibody, 1/200. 

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