How do you “Fractionate” a Cell?

cell-fractionation-v19

Different scientific questions focus on different parts of the cell and it is often necessary to break a cell up into those different pieces (figure above). While various “-omic” methods are well suited to answering global/systems-level questions for the four catagories listed above (e.g. microscopy, genomics, proteomics, metabolomics) they often lack the resolution of fractionation-methods to answer molecular level questions.

Organelle Fractionation relies on serial centrifugation at increasing speeds and is especially useful for studying the localization of proteins in the various compartments of the cell. For example, the sequestration of transcription factors in the cytosol is a common method to keep them from affecting gene expression in the nucleus.

DNA/RNA Fractionation is the foundation of most sequencing and PCR-based methods in molecular biology. As a chemical, DNA is very stable and easy to isolate for sequencing and PCR. RNA, on the other hand, is 107x less stable (see post on Timescales in Biology) and VERY susceptible RNAses and thus requires a specialized guanidinium-phenol-chloroform extraction method to isolate (see figure above). The percentages listed in DNA/RNA column represent typical eukaryotic dry-weight percentages of protein(60%), lipids(13%), DNA(1%) and RNA(4%) that can be separated via this method.2

Protein makes up the majority of a cell’s “machinery” and requires relatively gentile chromatographic methods to prevent “machine-breakage.” Affinity-chromatography, is the method of choice for protein-purification as a single column can purify a protein 1000x or more. Non-specific chromotographic methods, such as size-exclusion chromatography, on the other hand are less effective and often require serialization in order to obtain protein of sufficient purity for mechanistic study. The percentages listed in Protein column represent the functional-distribution of typical eukaryotic protein expression levels.2

Finally, metabolites are the raw materials that provide energy and materials for every process that occurs within a cell. As small organic molecules, studies on metabolites often rely on serial extraction with organic solvents. For example, when cell-lysate is exposed to a water-chloroform mixture, the fats partition to chloroform while sugars and salts partition to the water. The boxes listed in the Metabolite column above, represent typical metabolite concentrations as measured in bacterial cells undergoing exponential growth.2

 

REFERENCES:

  1. Alberts, B. Molecular Biology of the Cell 5th Ed. Garland Science 2008
  2. Milo, R.; Phillips, R. Cell Biology by the Numbers Garland Science 2015
  3. Green, M.R.; Sambrook, J. Molecular Cloning: A Laboratory Manual 4th Ed., 2012, Cold Spring Harbor Laboratory Press
  4. Murphy, K. Janeway’s Immunobiology 8th Ed. Garland Science 2012

 

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