Purifying organelles and analyzing their contents are fundamental in proteomic and metabolic research

The fractionation of organelles and sub-cellular fractionation offers many advantages:

  • Reduced complexity while maintaining biological context
  • Unmatched information about organelle specific protein functionality
  • Gives access to sub proteomes
  • Opens a new dimension essential to assess pathological altered organelles and their profiles
  • Adds confidence to your functional disease studies by removal of contaminants

1. Purification of mitochondria from Saccharomyces cerevisiae

The example below shows the homogenity and overall structural integrity of the ZE-FFE purified mitochondria. It is assessed by electron microscopy analysis of ultra thin slices of pelleted mitochondial preparations

mitochondria ZE 01

A cell homogenate of Saccharomyces cerevisiae was pre-purified by differential centrifugation. The electron microscopy image shows mitochondria, debris and contamination of the sample.

In a second step the sample was separated by Free Flow Electrophoresis in zone electrophoresis mode, to prify the mitochondria.

The mitochondria fractions were detected by the light scattering profil, measuring the optical density at 420 nm.

The peak at Fraction 41 was selected for further analysis.

This fraction revealed highest purity of intact mitochondria.

The native separation of intact mitochondria maintains the biological context and allows for further functional studies.

A high degree of purity could be achieved by the removal of contaminating organelles e.g. lysosomes and damaged mitochondria.

Also a significant decrease of degraded proteins due to the removal of endogenous cellular proteases was recorded.

A high sample load is enabled by continuous sample application which allows the separation of preparative amounts within short time.

This work is published by: Zischka et al. Proteomics 2003, 3, 906-916. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

 

 

 

 

 

 

 

 

 

 


 2. Identification of subpopulations of rat liver Peroxisomes

The example below shows the identification of two different subpopulations of Peroxisomes derived from rat liver homogenates. They are documented by electron microscopy analysis and enzymatic (Catalase) activity tests.

The rat liver homogenate was pre-purified by density gradient centrifugation and the pelleted sample was solubilized in FFE buffer.

The FFE separation was performed in zone electrophoresis mode and two different subpopulations of peroxisomes could be identified.

 peroxisomes ZE 01

The different populations revealed different enzymatic activity of Catalase.

This work was done in cooperation with Prof. Völkl and Dr. Islinger, Univ. Heidelberg. Reproduced with permission.