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

13 product images

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

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  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 (Anti-Alpha-synuclein antibody [MJFR1] ab138501). Anti-Alpha-synuclein antibody [MJFR1] 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 Anti-Alpha-synuclein antibody [MJFR1] ab138501 at 1/10 000 dilution and Anti-alpha Tubulin antibody [DM1A] - Loading Control 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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed 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).

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  Western blot - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (ab209420)

  Lanes 1 - 4: Merged signal (red and green). Green - Anti-Alpha-synuclein antibody [MJFR1] ab138501 observed at 14 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  

  Anti-Alpha-synuclein antibody [MJFR1] 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.  Anti-Alpha-synuclein antibody [MJFR1] ab138501 and Anti-GAPDH antibody [6C5] - Loading Control 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 Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed 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 (Anti-Alpha-synuclein antibody [MJFR1] ab138501).

  All lanes: Western blot - Anti-Alpha-synuclein antibody [MJFR1] (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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  Immunoprecipitation - Anti-Alpha-synuclein antibody [MJFR1] - BSA and Azide free (ab209420)

  alpha Synuclein was immunoprecipitated from Human fetal brain tissue using purified Anti-Alpha-synuclein antibody [MJFR1] ab138501 at 1/600 dilution. Western blot was performed from the immunoprecipitate using purified Anti-Alpha-synuclein antibody [MJFR1] 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 (Anti-Alpha-synuclein antibody [MJFR1] ab138501).

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

  Predicted band size: 14 kDa

  Observed band size: 18 kDa

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  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 Anti-Alpha-synuclein antibody [MJFR1] 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 (Anti-Alpha-synuclein antibody [MJFR1] ab138501, 1μg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) presorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG isotype control antibody (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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 (Anti-Alpha-synuclein antibody [MJFR1] ab138501).

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  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

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

  Lanes 1 - 4: Merged signal (red and green). Green - Anti-Alpha-synuclein antibody [MJFR1] ab138501 observed at 18 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 37 kDa.

  Anti-Alpha-synuclein antibody [MJFR1] ab138501 was shown to react with Alpha-synuclein in wild-type HEK-293T. Loss of signal was observed when knockout cell line Human SNCA (Alpha-synuclein) knockout HEK-293T cell line ab255433 (knockout cell lysate Human SNCA (Alpha-synuclein) knockout HEK-293T cell lysate ab263769) was used. Wild-type and Alpha-synuclein knockout samples were subjected to SDS-PAGE. Anti-Alpha-synuclein antibody [MJFR1] ab138501 and Anti-GAPDH antibody [6C5] - Loading Control (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (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

  Performed under reducing conditions.

  Predicted band size: 14 kDa

  Observed band size: 14 kDa

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  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 PE Anti-Alpha-synuclein antibody [MJFR1] ab209306 for protocol details.
  

  Overlay histogram showing Neuro 2A (differentiated) cells stained with PE Anti-Alpha-synuclein antibody [MJFR1] 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 (PE Anti-Alpha-synuclein antibody [MJFR1] ab209306, 1/2500 dilution) for 30 min at 22°C.

  Isotype control antibody (black line) was rabbit IgG (monoclonal) Phycoerythrin (PE Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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.

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

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

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  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

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  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 Alexa Fluor® 488 Anti-Alpha-synuclein antibody [MJFR1] ab195025 for protocol details.
  

  Overlay histogram showing Neuro 2A cells stained with Alexa Fluor® 488 Anti-Alpha-synuclein antibody [MJFR1] 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 (Alexa Fluor® 488 Anti-Alpha-synuclein antibody [MJFR1] ab195025, 1/100 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor^® 488 (Alexa Fluor® 488 Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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.

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

  Immunohistochemical analysis of formalin fixed paraffin embedded human cerebrum labelling Alpha-synuclein with Anti-Alpha-synuclein antibody [MJFR1] 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.

  Anti-Alpha-synuclein antibody [MJFR1] 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).