Jun 13, 2023

Public workspaceWestern Blot Analysis V.1

DOI
dx.doi.org/10.17504/protocols.io.j8nlkwp25l5r/v1
  • 1Mitochondria and Inflammation in Neurodegenerative Diseases, DZNE, Tübingen-Germany;
  • 2Hertie Institute for Clinical Brain Research, University of Tübingen;
  • 3Neuropharmacology Laboratory, Oasi Research Institute-IRCCS, Troina, Italy;
  • 4Biomedical and Biotechnological Sciences, Pharmacology Section, University of Catania-Italy
carmela giachino
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkwp25l5r/v1
Protocol Citation: michela.deleidi, Bianca Marchetti, Federico Bertoli, Carmela Giachino 2023. Western Blot Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkwp25l5r/v1
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: March 31, 2023
Last Modified: June 13, 2023
Protocol Integer ID: 79825
Keywords: Western Blot Analysis
Funders Acknowledgement:
ASAP-Aligning Science Across Parkinson’s
Grant ID: ASAP-000420
Abstract
Western Blotting is a technique for the immunodetection of proteins using antibodies with fluorescent or chemiluminescent detection.
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Materials
Materials

  • 0.33 M sucrose
  • 8 mM Hepes, pH 7.4
  • Laemli SDS boiling buffer (Sigma)
  • 9-12% SDS-polyacrilamide gel
  • polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ)
  • 5% non-fat dry milk
  • TBST

Western Blot Analysis
Western Blot Analysis
4h 40m
4h 40m
Prepare protein extracts as previously described.
Homogenize tissues in lysis buffer (Concentration0.33 Molarity (M) sucrose/Concentration8 millimolar (mM) Hepes, Ph7.4 and protease inhibitors) and quantify them using the BCA protein determination method (Bio-Rad, Hercules, CA).

Dilute protein samples to equivalent volumes containing Amount20 µg of protein and boil in an equal volume of Laemli SDS boiling buffer (Sigma) for Duration00:10:00 .

10m
Load samples into a 9-12% SDS-polyacrilamide gel and separate it by electrophoresis for Duration03:00:00 at 100 V.

3h
Transfer the proteins to polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ) for Duration01:30:00 at 300 mA.

1h 30m
After blocking of nonspecific binding with 5% non-fat dry milk in TBST, probe the membranes with primary antibodies and process.
Perform densitometric analysis using ImageQuantity One.
Analyze
Normalize data to β-actin, normalize values of phosphorylated GSK-3β (pTyr216 GSK-3β); phosphorylated α-syn (pSer129 α-syn) and phosphorylated tau (pSer396 tau) to total GSK-3β, α-syn, and tau, respectively, before statistical analysis of variance and express values as percent changes (%) of WT controls.

Dashed lines (in white) indicate discontinuous bands (nonsequential lanes) taken from the same blot, at the same molecular weight (mass – kDa) in order to better represent the mean signal from all values (5-6 individual blots/genotype/treatment) for that particular group. Corresponding control bands (loading controls) match experimental bands.