All lanes : Anti-Alpha-synuclein antibody [MJFR1] (ab138501) at 1/10000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : SNCA knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

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

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.

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.

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).

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).

All lanes : Anti-Alpha-synuclein antibody [MJFR1] (ab138501) at 1/10000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : SNCA knockout HAP1 whole cell lysate
Lane 3 : Human brain whole cell lysate
Lane 4 : Mouse brain whole cell lysate

Lysates/proteins at 40 µg per lane.

Predicted band size: 14 kDa

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.

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.

Anti-Alpha-synuclein antibody [MJFR1] (ab138501) at 1/10000 dilution (purified) + Human fetal brain at 20 µg

Secondary
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 why is the actual band size different from the predicted?

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

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).

Immunocytochemistry/Immunofluorescence analysis of U87-MG (Human glioblastoma-astrocytoma epithelial cell line) cells labeling alpha Synuclein with unpurified ab138501 at 1/15 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 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 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

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.

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.

alpha Synuclein was immunoprecipitated from Human fetal brain tissue using unpurified ab138501 at 1/50 dilution. Western blot was performed from the immunoprecipitate using unpurified 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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human clear cell carcinoma of kidney labeling alpha Synuclein with unpurified ab138501 at 1/15 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human brain tissue labeling alpha Synuclein with unpurified ab138501 at 1/300 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Flow cytometry analysis of 2% paraformaldehyde fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling alpha Synuclein with unpurified ab138501 at 1/20 dilution. The secondary antibody was Goat anti rabbit IgG (FITC) at 1/150 dilution.

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