Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free

• IHC

Lab

Immunohistochemistry - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

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

• WB

Lab

Western blot - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

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 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138501).

All lanes:

Western blot - Anti-Alpha-synuclein antibody [MJFR1] (<a href='/en-us/products/primary-antibodies/alpha-synuclein-antibody-mjfr1-ab138501'>ab138501</a>) 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

Lab

Western blot - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

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 18 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab138501 was shown to react with Alpha-synuclein in wild-type HEK-293T. Loss of signal was observed when knockout cell line ab255433 (knockout cell lysate ab263769) was used. Wild-type and Alpha-synuclein knockout samples were subjected to SDS-PAGE. ab138501 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 dilution and 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.

All lanes:

Western blot - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (ab209420) at 1/10000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

SNCA knockout HAP1 cell lysate at 20 µg

Lane 3:

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

Lane 4:

SNCA knockout HEK-293T cell lysate at 20 µg

Predicted band size: 14 kDa

Observed band size: 14 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138501).

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

IHC image of Alpha synuclein staining in a section of frozen normal human Parkinson's brain* performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab209420, 0.5ugml, 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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

Negative control image : IHC image of Alpha synuclein staining in a section of frozen normal human liver* performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab209420, 0.5ugml, 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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138501).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

This data was developed using the same antibody clone in a different buffer formulation (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 1/10 000 dilution 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)

Unknown

Flow Cytometry (Intracellular) - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

Clone MJFR1 (ab209420) has been successfully conjugated by Abcam. This image was generated using Anti-Alpha-synuclein antibody [MJFR1] (Alexa Fluor^® 488). Please refer to ab195025 for protocol details.

Overlay histogram showing Neuro 2A cells stained with ab195025 (red line). The cells were fixed with 4% formaldehyde (10 min) then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. Cells were then incubated with the antibody (ab195025, 1/100 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor^® 488 (ab199091) used at the same concentration and conditions as the primary antibody. 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.

This antibody gave a positive signal in Neuro 2A cells fixed with 80% methanol (5 min) used under the same conditions.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

Clone MJFR1 (ab209420) has been successfully conjugated by Abcam. This image was generated using Anti-Alpha-synuclein antibody [MJFR1] (PE). Please refer to ab209306 for protocol details.

Overlay histogram showing Neuro 2A (differentiated) cells stained with ab209306 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min at 22°C. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab209306, 1/2500 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138501).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138501).

• IP

Supplier Data

Immunoprecipitation - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (AB209420)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab138501).

All lanes:

Immunoprecipitation - Anti-Alpha-synuclein antibody [MJFR1] (<a href='/en-us/products/primary-antibodies/alpha-synuclein-antibody-mjfr1-ab138501'>ab138501</a>)

Predicted band size: 14 kDa

Observed band size: 18 kDa

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