Anti-Alpha-synuclein antibody [MJFR1]

• WB

Lab

Western blot - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

This data was developed using the same antibody clone in a different buffer formulation (ab138501).

Lanes 1- 2 : Merged signal (red and green). Green - ab138501 observed at 18 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab138501 was shown to react with SNCA in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255433 (knockout cell lysate ab263769) was used. Wild-type HEK-293T and SNCA knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab138501 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lanes 1 - 2:

Western blot - Anti-Alpha-synuclein antibody [MJFR1] (ab138501) at 1/10000 dilution

Lanes 1 - 2:

Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (<a href='/en-us/products/unavailable/alpha-synuclein-antibody-mjfr1-bsa-and-azide-free-ab156369'>ab156369</a>) at 1/10000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

SNCA knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human SNCA (Alpha-synuclein) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-snca-alpha-synuclein-knockout-hek-293t-cell-line-ab255433'>ab255433</a>)

Predicted band size: 14 kDa

Observed band size: 18 kDa

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• IHC

Lab

Immunohistochemistry - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

Immunohistochemical analysis of formalin fixed paraffin embedded human cerebrum labelling Alpha-synuclein with ab138501 at a concentration of 0.01 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC2) 100°C, pH 8.5 for 32 mins.

ab138501 anti-Alpha-synuclein [MJFR1] was incubated for 16 mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

ab138501 staining Alpha-Synuclein in wild-type Hap1 cells (top panel) and SNCA knockout Hap1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab138501 at 0.1 μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

Intracellular Flow Cytometry analysis of 2% paraformaldehyde fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling alpha Synuclein with purified ab138501 at 1/200 dilution. The secondary antibody was Goat anti rabbit IgG (FITC) at 1/150 dilution.

The Isotype control is Rabbit monoclonal IgG (green line).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

Immunocytochemistry/Immunofluorescence analysis of U87-MG (Human glioblastoma-astrocytoma epithelial cell line) cells labeling alpha Synuclein with purified ab138501 at 1/150 dilution (left panel). Cells were fixed with 4% paraformaldehyde. A Goat anti rabbit IgG(Alexa Fluor^®555) (1/200) was used as the secondary antibody. DAPI (blue) was used as the nuclear counter stain (right panel).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

IHC image of alpha Synuclein staining in Normal human Substantia Nigra formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab138501, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human clear cell carcinoma of kidney labeling alpha Synuclein with purified ab138501 at 1/150 dilution. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Prediluted HRP Polymer for Rabbit IgG was used as the secondary antibody. Counter stained with Hematoxylin.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

IHC image of alpha Synuclein staining in Parkinson Human Substantia Nigra formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab138501, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-SNCA knockout cells (red line) stained with ab138501. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab138501, 1μg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-SNCA knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

IHC image of alpha Synuclein staining in normal Human cerebral cortex formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab138501, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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.

• IP

Supplier Data

Immunoprecipitation - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

alpha Synuclein was immunoprecipitated from Human fetal brain tissue using purified ab138501 at 1/600 dilution. Western blot was performed from the immunoprecipitate using purified ab138501. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as secondary antibody at 1/1000 dilution. Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Alpha-synuclein antibody [MJFR1] (ab138501)

Predicted band size: 14 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

Lanes 1 - 4 : Merged signal (red and green). Green - ab138501 observed at 14 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab138501 was shown to specifically react with SNCA in wild-type HAP1 cells. No band was observed when SNCA knockout samples were used. Wild-type and SNCA knockout samples were subjected to SDS-PAGE. ab138501 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10000 and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Alpha-synuclein antibody [MJFR1] (ab138501) at 1/10000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 40 µg

Lane 2:

SNCA knockout HAP1 whole cell lysate at 40 µg

Lane 3:

Human brain whole cell lysate at 40 µg

Lane 4:

Mouse brain whole cell lysate at 40 µg

Predicted band size: 14 kDa

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• WB

Supplier Data

Western blot - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Alpha-synuclein antibody [MJFR1] (ab138501) at 1/10000 dilution

All lanes:

Human fetal brain at 20 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 14 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Anti-Alpha-synuclein antibody [MJFR1] (AB138501)

This data was developed using the same antibody clone in a different buffer formulation (ab138501). **Lanes 1 - 4 : ** Merged signal (red and green). Green - ab138501 observed at 14 kDa. Red - loading control, ab8245, observed at 37 kDa. ab138501 was shown to specifically react with SNCA in wild-type HAP1 cells. No band was observed when SNCA knockout samples were used. Wild-type and SNCA knockout samples were subjected to SDS-PAGE.  ab138501 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10000 and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Alpha-synuclein antibody [MJFR1] (ab138501) at 1/10000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20.0 µg

Lane 2:

SNCA knockout HAP1 whole cell lysate at 20.0 µg

Lane 3:

Human brain whole cell lysate at 20.0 µg

Lane 4:

Mouse brain whole cell lysate at 20.0 µg

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