May 14, 2024

Aggregated aSyn Dot Blot assay

 Forked from Aggregated aSyn Dot Blot assay
DOI
https://dx.doi.org/10.17504/protocols.io.bp2l629x1gqe/v1
  • 1California Institute of Technology
gustavo.parfitt Parfitt
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l629x1gqe/v1
External link: https://doi.org/10.7554/eLife.81453
Protocol Citation: rabdelha 2024. Aggregated aSyn Dot Blot assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l629x1gqe/v1
Manuscript citation:
Abdel-Haq R, Schlachetzki JC, Boktor JC, Cantu-Jungles TM, Thron T, Zhang M, Bostick JW, Khazaei T, Chilakala S, Morais LH, Humphrey G, Keshavarzian A, Katz JE, Thomson M, Knight R, Gradinaru V, Hamaker BR, Glass CK, Mazmanian SK, A prebiotic diet modulates microglial states and motor deficits in α-synuclein overexpressing mice. eLife doi: 10.7554/eLife.81453
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 14, 2024
Last Modified: May 31, 2024
Protocol  Integer ID: 99796
Keywords: Aggregated aSyn Dot Blot assay, Proteinase K resistant aSyn histology, ASAPCRN, asyn dot blot assay, aggregated asyn dot blot, assay, protocol detail, protocol
Abstract
This protocol details aggregated aSyn Dot Blot assay.
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Aggregated aSyn Dot Blot assay
Normalize protein samples to equal concentrations between 100 ng -1000 µL in PBS.

Carefully spot 1.5 µL each sample onto dry nitrocellulose (0.45 µm pores) membrane.

Allow moisture to dry for a minute or two.
Block membrane in 5% skim milk in TBST.
Wash membrane 1X in TBST.
Apply anti-aggregated aSyn antibody in 5% skim milk in TBST (1:1000 for Abcam fibril.aggregate specific aSyn antibody: MJFR 14-6-4-2).
Incubate at Room temperature for 1-2 hours, or Overnight at 4 °C .

1h
Wash 3x with TBST.
Wash with TBST (1/3).
Wash with TBST (2/3).
Wash with TBST (3/3).
Incubate with anti-rabbit IgG (or appropriate secondary)at 1:1000 for 1-2 hours in TBST.
Wash 3x with TBST.
Wash with TBST (1/3).
Wash with TBST (2/3).
Wash with TBST (3/3).
Develop with Biorad Chemiluminescent substrate, or similar.
Proteinase K resistant aSyn histology
1h 10m
Note
Certainly can be modified for either DAB or Fluorescent staining, I have DAB protocol too

Wash 50um brain sections 1x in PBS.
Treat with Protinase K (NEB, 1:4000, stock at 20 mg/mL ) for 00:10:00 at Room temperature .

10m
Wash 2x with IHC buffer (3% NGS, 0.3% TX100, in PBS).
Wash with IHC buffer (3% NGS, 0.3% TX100, in PBS) (1/2).
Wash with IHC buffer (3% NGS, 0.3% TX100, in PBS) (2/2).
Block with Mouse-on-Mouse kit (Vector Lab, PK-2200, just first step in instructions) for 01:00:00 .

1h
Wash 2x with IHC buffer.
Wash with IHC buffer (1/2).
Wash with IHC buffer (2/2).
Incubate Overnight at 4 °C with primary antibody 1:250 – 1:1000 (anti-synuclein BD Biosci 610787).

Wash 3x with PBS.
Wash with PBS (1/3).
Wash with PBS (2/3).
Wash with PBS (3/3).
Incubate with desired secondary (anti-Mouse IgG AF488 is my choice at 1:1000).
Wash 3x with PBS.
Wash with PBS (1/3).
Wash with PBS (2/3).
Wash with PBS (3/3).
Stain with Nissl and Mount with Profade with DAPI.