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Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics
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Biochemistry
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JoVE Journal Biochemistry
Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

DOI:
10.3791/58581-v

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10:28 min

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October 19, 2018

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, , , ,
1Université Grenoble Alpes, INRA, CNRS, CEA

Chapters

  • 00:04Title
  • 01:01Harvesting of Arabidopsis Leaves
  • 02:26Purification of Crude Chloroplasts Using Differential Centrifugation
  • 03:30Purification of Intact Chloroplasts on Continuous Percoll Gradient
  • 04:51Lysis of Intact Chloroplasts Using a Hypotonic Buffer and Purification of Chloroplast Sub-Compartments on Discontinuous Sucrose Gradients
  • 06:05Washing and Concentration of Thylakoid and Envelope Membrane Systems
  • 07:09Results: Analysis of Protein Localization by Immunoblotting or Proteomics
  • 08:44Conclusion

Summary

Automatic Translation

Automatic Translation

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Tags

Keywords: Chloroplast Sub-compartmentsProtein LocalizationImmunoblottingProteomicsArabidopsisChloroplast FractionationChloroplast IsolationChloroplast PurificationSubcellular FractionationChloroplast EnvelopeChloroplast StromaChloroplast Thylakoid Membranes

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