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Methods:Polyethyleneimine precipitation

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Purpose

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Polyethyleneimine is used to precipitate nucleic acids from crude cell lysates. Depending on the salt concentrations used, this can be used to purify nucleic acid binding protein along with the pellet, or remove DNA and RNA from the supernatant. Polyethylineimine is also known as PEI or Polymin P.

Being a highly positively charged polymer, at neutral pH PEI interacts strongly with nucleic acids, which are negatively charged polyelectolytes. PEI precipitation is basically a titration, so the amount needed depends on the amount of material in the extract, not on the solubility properties of the material.

Materials Needed

Polyethyleneimine is usually purchased in solution and kept as a 5% w/vol stock solution at pH7.9. PEI is typically purchased as mixed polymers of MW 30-90K.

Protocol

Add PEI solution slowly with mixing to the desired final concentration, centrifuge, and collect the supernatant or pellet as desired. When added to a lysate, the solution rapidly becomes milky and opaque. Precipitation is rapid, but the suspension can be kept on ice for a few hours.

Burgess [1] reviewed the use of PEI in protein purification, dividing the applications into three strategies:

High ionic strength

At 1M NaCl, nucleic acids are removed, and almost no proteins remain bound to the pellet. This is not used because

  • it offers little purification of the desired protein
  • the resultant supernatant is too salty to use for many subsequent chromatographic methods
  • excess PEI will precipitate proteins from the supernatant when it is diluted to lower ionic strength.

Medium ionic strength

At ionic strength between 0.2-1M NaCl, nucleic acids and some proteins will be precipitated. The desired protein remains in the supernatant.

Low ionic strength

Between 0.1-0.2M NaCl, many nucleic acid binding proteins remain bound to the nucleic acid, which is precipitated by PEI. The desired protein is recovered from the pellet at higher ionic strength.

Removal of PEI

The PEI that remains in solution can be a problem for subsequent steps. Note that PEI does not have significant UV absorbance, so it is not straightforward to measure the PEI remaining. A protein of interest can sometimes be removed from the PEI by ammonium sulfate precipitation, or PEI can be removed from the solution by passing it over a negatively charged chromatography medium, such as phosphocellulose or CM-sepharose.

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References

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  1. Burgess, RR (1991) Use of polyethyleneimine in purification of DNA-binding proteins. Meth. Enzymol. 208 3-10 PubMed EcoliWiki page