Total protein was extracted from cell pellets using RIPA Lysis buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% DOC, 1% NP-40, 0.1% SDS, and proteinase inhibitor cocktail). Protein extracts were used for the DC Protein Assay (Bio-Rad), and 7.5 μg of total protein extracts per sample were subjected to native dot blot analyses as previously described64 (link). Total protein staining Revert 700 Stain Solution (Li-COR) was used as a loading control. Antibody signal intensities were normalized to the total protein. Antibodies used for the blotting analysis were as follows: primary antibody MJFR-14-6-4-2 (1:2000 #ab209538; Abcam) overnight at 4 °C and secondary antibody IRDye 800CW Goat anti-Rabbit (1:20000; Li-COR) for 1 h at room temperature. Detection and digitalization were performed using Odyssey CLx Infrared Imaging System (Li-COR). All the native dot blots originated from the same experiment and were processed in parallel.
Mjfr 14 6 4 2
Manufactured by Abcam
Sourced in United Kingdom
The MJFR-14-6-4-2 is a lab equipment product. It is a device designed for use in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Ab209538, by Abcam (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Irdye 800cw donkey anti rabbit secondary antibody, by LI COR (1 mentions)
Direct blue 71, by Merck Group (1 mentions)
Biotek synergy h1 multimode reader, by Agilent Technologies (1 mentions)
96 well plate, by Corning (1 mentions)
Anti α syn c20, by Santa Cruz Biotechnology (1 mentions)
Anti flotillin 1, by Santa Cruz Biotechnology (1 mentions)
Anti αsyn syn 1, by BD (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
4 protocols using mjfr 14 6 4 2
1
Native Dot Blot Analysis of Protein Extracts
2
Native Dot Blot Analysis of aSyn
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Native dot blot analysis of aggregated aSyn was performed by applying the cell lysate containing 10 µg total protein on a nitrocellulose membrane (0.45 µm, Buckinghamshire, UK) in a total volume of 5 µL. Membrane was air-dried for 3 h and subsequently blocked in 5% non-fat dry milk in TBS for 1 h at RT. Aggregated aSyn was probed by using a rabbit conformation-specific antibody MJFR-14-6-4-2 (Abcam, Cambridge, UK) in combination with IRDye 800CW donkey anti-rabbit secondary antibody (LI-COR Biosciences, Lincoln, NE, USA) using the identical immunostaining protocol for WB. Fluorescent signals were detected by using the Odyssey imaging system (LI-COR Biosciences). Loading of total protein was controlled by staining total protein loaded using direct blue 71 according to Hong et al. [53 ]. For this purpose, the blot membrane was stained by using a working solution of direct blue 71 containing 0.008% direct blue 71 (Sigma Aldrich), 40% Ethanol and 10% acetic acid for 5 min at RT, followed by rinsing the membrane with 40% ethanol and 10% acetic acid.
3
Quantification of α-Synuclein Aggregates
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For the measurement of α-synuclein aggregates, 96-well plates (Corning Costar, Glendale, AZ, USA) were coated overnight at 25 °C with 1 μg/mL of the mouse monoclonal antibody Syn-F2 (50 μL/well) in 100 mM NaHCO3 (pH 9.6). The plates were washed three times with wash buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween-20). Standards and samples were diluted in 10 mM Tris-Cl, pH 7.6, 100 mM NaCl, 0.1% Tween-20 and 1% BSA (TBS-T/BSA buffer) and incubated for 2.5 h at 37 °C. After washing three times, the rabbit monoclonal antibody MJFR-14-6-4-2 (Abcam, Cambridge, UK) was added at a concentration of 74 ng/mL and incubated for 1 h at 25 °C. The wells were washed again three times, and the anti-rabbit IgG-HRP antibody (Agilent, Santa Clara, CA, USA) was applied at a concentration of 16.7 ng/mL (1:15,000 dilution in TBS-T/BSA buffer) for 30 min at 4 °C. The bound HRP was detected by adding 50 μL/well of HRP substrate (Luminata Crescendo ELISA HRP chemiluminescent substrate, Merck Millipore), and the chemiluminescence in relative light units was measured after 5 min in a Biotek Synergy H1 multimode reader (Agilent, Santa Clara, CA, USA).
4
Immunoblotting of alpha-synuclein in fractions
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Fifty μg of sucrose gradient purified fractions (B, C, and D), diluted in PBS, were sonicated for 5 min in a water-bath sonicator, and run on a 13% SDS-PAGE Tris-glycine gel. Proteins were subsequently transferred onto nitrocellulose membranes (Amersham, 106000001) and analyzed by immunoblotting. For dot blot analysis, 20 μg of pre-sonicated fractions C were loaded on a multi-well device, and proteins were bound to a nitrocellulose membrane (Amersham, 10600001), under vacuum for 30 min at RT. Membranes were blocked in blocking buffer (5 % non-fat milk, 0.05% Tween-20 in TBS), for 1 h at RT and probed overnight at 4oC with the following primary antibodies: anti-α-Syn C20 (Santa Cruz, 1:1000), anti-α-syn Syn1 (BD biosciences, 1:1,000), anti-pS129 α-Syn (Abcam, 1:1,000), anti-Flotillin-1 (Santa Cruz, 1:1,000), and recombinant anti-α-Syn aggregate antibody (conformation-specific MJFR-14-6-4-2, Abcam, 1:50,000). Following 10 min washes with 0.05% Tween-20 in TBS (TBS-T) thrice, secondary antibodies (Merck-Millipore) diluted in blocking buffer were added for 2 h at RT. Membranes were rinsed again as above and Clarity Western ECL Substrate (Bio-Rad) was used for the detection of the proteins bound to nitrocellulose membranes (Supplementary Table 6 ).
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