Immunoblot Analysis of α-Synuclein in Transgenic Mice

Hemibrains were homogenized and divided into cytosolic and membrane fractions as previously described [54 (link),55 (link)]. For immunoblot analysis, 20 μg of total protein per lane was loaded on 4-12% Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride membranes. To determine the effects of the immunotherapy in levels of α-syn, blotted samples from immunized α-syn tg mice were probed with antibodies against full length human α-syn (1:1000, SYN211, Life Technologies). Incubation with primary antibody was followed by species-appropriate incubation with secondary antibody tagged with horseradish peroxidase (1:5000, Santa Cruz Biotechnology), visualization with enhanced chemiluminescence, and analysis with a Versadoc XL imaging apparatus (BioRad). Analysis of β-actin (Sigma) levels was used as a loading control.
For studying which species the antibodies elicited by AFF 1 recognize, recombinant or 4-hydroxy-2-nonenal-treated α-syn [25 (link)] were loaded on 4-12% Bis-Tris SDS-PAGE gels and analyzed by immunoblot using AFF 1-elicited antibodies as primary antibody. The monoclonal antibody LB509 (Covance) served as positive control.

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Mandler M., Valera E., Rockenstein E., Mante M., Weninger H., Patrick C., Adame A., Schmidhuber S., Santic R., Schneeberger A., Schmidt W., Mattner F, & Masliah E. (2015). Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy. Molecular Neurodegeneration, 10, 10.

Publication 2015

SYN211 horseradish peroxidase Versadoc XL imaging apparatus β-actin LB509

4 hydroxy 2 nonenal Actin Antibodies Antibody Bis tris Chemiluminescence Cytosolic Gels Horseradish peroxidase Human Immunoblot Immunotherapy Membranes Mice Monoclonal antibody Polyvinylidene fluoride Protein Sds page Syn 1

Corresponding Organization : University of California, San Diego

Other organizations : AFFiRiS (Austria), Vienna Biocenter

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Variable analysis

independent variables

Immunotherapy (immunized α-syn tg mice)

dependent variables

Levels of α-syn
Species of α-syn recognized by AFF 1-elicited antibodies

control variables

Total protein loaded per lane (20 μg)
4-12% Bis-Tris SDS-PAGE gels
Polyvinylidene fluoride membranes for blotting
Primary antibody dilution (1:1000 for SYN211, Life Technologies)
Secondary antibody dilution (1:5000 for Santa Cruz Biotechnology)
β-actin levels as loading control
Recombinant or 4-hydroxy-2-nonenal-treated α-syn as samples for studying species recognized by AFF 1-elicited antibodies

positive control

Monoclonal antibody LB509 (Covance) as positive control for studying species recognized by AFF 1-elicited antibodies

negative control

Not explicitly mentioned

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