α-Synuclein preparations were separated on SDS-PAGE gels and transferred onto 0.45 μm nitrocellulose membranes at 100 volts for 30 min. Membranes were blocked with PBS containing 5% non-fat dry milk and probed with α-synuclein antibody (1:1000, mouse monoclonal, BD biosciences, North Ryde, Australia) overnight at 4 °C. They were then washed three times in PBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody (1:2000, Dako, Carpinteria, CA, USA) for 2 h. Signals were detected using enhanced chemiluminescence and X-ray films (ECL, GE Healthcare, Buckinghamshire, UK).
α synuclein antibody
Manufactured by BD
Sourced in Australia
The α-synuclein antibody is a laboratory reagent used for the detection and quantification of the α-synuclein protein. α-synuclein is a small protein found predominantly in the presynaptic terminals of neurons and is associated with various neurodegenerative disorders. The antibody can be used in techniques such as Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) to study the expression and localization of α-synuclein in biological samples.
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Lab products found in correlation
Criterion stain free 4 20 sds page gels, by Bio-Rad (2 mentions)
Nfl antibody, by Cell Signaling Technology (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Gel doc system, by Bio-Rad (2 mentions)
Mouse monoclonal, by BD (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Protein a magnetic bead, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
C2 confocal microscope, by Nikon (1 mentions)
6 protocols using α synuclein antibody
1
Western Blot Analysis of α-Synuclein
2
Quantitative Analysis of Neuronal Proteins
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Protein lysates (10 µg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris–HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4–20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 V for 30 min. The membranes were blocked with TBS containing 5% nonfat dry milk and probed with α-synuclein antibody (1:1000, BD Biosciences, 610,787), NFL antibody (1:2000, Cell Signaling, 2835S) or acrolein antibody (1:1000, NOVUS, NB200-556) overnight at 4 °C. They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The blots were stripped and probed for housekeeper proteins tubulin or GAPDH. The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45 s). ELISA of α-synuclein (Legend Max Cat no: 844101) was carried out following the manufacturer’s protocol. ELISA of neurofilament light (Novus Cat no: NBP2-81,184) was carried out following the manufacturer’s protocol.
3
Antibody Sourcing for Synaptic Proteins
4
Western Blot Analysis of α-Synuclein and NfL
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Protein lysates (15 µg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris-HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4-20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 volts for 30 min. The membranes were blocked with TBS containing 5% nonfat dry milk and probed with α-synuclein antibody (1:1000, BD Biosciences, 610787) or NfL antibody (1:2000, Cell Signaling, 2835 S) overnight at 4 °C. They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The blots were stripped and probed for housekeeper proteins GAPDH or β-actin. The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45 s). All blots were derived from the same experiment and that they were processed in parallel. ELISA of plasma α-synuclein (Legend Max #844101) was carried out following the manufacturer’s protocol.
5
Immunoprecipitation of SNCA Fusion Proteins
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Bacterial cell lysates with IPTG-induced VN1–211-SNCA and SNCA-VC212–239 proteins or VN1–211-SNCA protein alone were prepared by sonication in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (pH 8.0), 1 mM ethylene glycol tetraacetic acid (pH 8.0), 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and a protease inhibitor cocktail (Sigma-Aldrich). After quantification, proteins (20 µg) were immunoprecipitated with rabbit green fluorescent protein (GFP) primary antibody (1 µg; Bioman Scientific Co. #HIT001R, Taipei, Taiwan) or normal immunoglobulin (IgG) antibody (as a negative control) (1 µg; Santa Cruz Biotechnology #sc-2027, Santa Cruz, CA, USA) conjugated to protein A magnetic beads (Millipore). The beads–proteins–antibody mixtures were washed three times with lysis buffer and the immunoprecipitated proteins were eluted by boiling in SDS sample loading buffer (50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue), separated on a 10% SDS-polyacrylamide gel and immunoblotting with α-synuclein antibody (1:1000; BD Biosciences #610787) as described.
6
Immunofluorescence Analysis of α-Synuclein
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OLN-AS7 cells grown in 8-well chamber slides were fixed with 4% paraformaldehyde. They were then permeabilized (1 x PBS, 0.25% Triton X-100), blocked (1 x PBS, 3% BSA) and incubated with α-synuclein antibody (BD Biosciences, #610787, 1 : 100) overnight at 4°C. Cells were washed with PBS prior to incubation with Alexa Fluor 488 fluorophore-conjugated goat anti-mouse antibody (Thermo Fisher, 3% BSA, 1 : 250) for 1 h. The cells were washed and counterstained with 4’,6-diamidino-2-phenylindole (DAPI, 1μl/ml in PBS) for 7 min. A coverslip was mounted to the slide using mounting media, and the cells were visualised with a confocal microscope (Nikon C2 Confocal Microscope). Ten randomly selected microscopic fields were captured for both control and CDH4-transfected cells at 20x magnification. Each field contained 10-30 cells. The relative α-synuclein intensity was calculated by dividing the total immunofluorescence quantity of α-synuclein by the cell count in each field using Image J Vl.47 software.
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