Apr 11, 2022

Public workspaceWestern blotting in Chlamydomonas reinhardtii V.2

PLOS One
DOI
dx.doi.org/10.17504/protocols.io.14egnjnpv5dy/v2
  • João Vitor Molino1
  • 1University of São Paulo
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DOI: dx.doi.org/10.17504/protocols.io.14egnjnpv5dy/v2
External link: https://doi.org/10.1371/journal.pone.0192433
Protocol Citation: João Vitor Molino 2022. Western blotting in Chlamydomonas reinhardtii. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnjnpv5dy/v2Version created by Joao Vitor Molino
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: April 11, 2022
Last Modified: April 11, 2022
Protocol Integer ID: 60613
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Abstract
This protocols describe the steps to perform a western blot in Chlamydomonas reinhardtii cell lysate and supernatant samples.
Guidelines
Prepare cell material by cultivating in liquid media. 
Before start
  • Check antibody dilutions
  • Check buffers disponibility
  • Prepare 5% milk solution
Sample preparation - Supernatant
Sample preparation - Supernatant
  1. Centrifuge algae culture at 2000xg for 10 min
  2. Recover supernatant 
Duration00:10:00 Centrifugation time
Sample preparation - Lysate
Sample preparation - Lysate
  1. Centrifuge algae culture at 2000xg for 10 min
  2. Remove supernatant, and re-suspend cells in lysis buffer (50 mM Tris·HCL (pH 8.0), 0.1% Triton X-100), concentrating cells 100-fold.
Duration00:10:00 Centrifugation time
Sonication
Sonication
  1. Sonicate using appropriate sonication tip. Duty cycle: 0.5 | Cycle: 1.0 s | Amplitude: 20% | Duration: 30 s
  2. Centrifuge for 15 min at 20000xg to remove cells debries and recover soluble proteins
  3. Quantificate soluble protein
Duration00:15:00 Centrifugation time
Gel electrophoresis | SDS-PAGE
Gel electrophoresis | SDS-PAGE
  1. Load 30 µg of total soluble protein (TSP) per lane in a 12% SDS-PAGE 
  2. Transfer proteins to a nitrocelulose membrane
  3. Block the membrane with 5% milk solution
  4. Probe the desired protein with the specific antibodie, diluted in 5% milk solution
  5. Wash 2 times with TBST (0.2 M Tris, 1.37 M NaCl, 0.1% Tween-20, pH 7.6)
  6. Add secondary antibody if required
Amount30 µg total solube protein per lane