Removal of Nucleic Acids
Polyethyleneimine (PEI) precipitation (pET CBD Expression System Manual - NOVAGEN)
Determine the amount of PEI necessary to clarify your lysate by performing
a titration of PEI on a series of aliquots of your sample. Add 0-140µl/ml
10% PEI (Polymin P of Sigma) to each aliquot and vortex briefly.
Usually 0.08% to 0.1% w/v is enough for a 10mg protein/ml solution at pH
7.5 to 8.0.
Spin for 2 minutes at 14000 rpm.
Load 5µg samples of each supernatant in PAGE-SDS gel and stain with Coomasie Blue.
The maximum level of PEI that does not precipitate your target protein can be applied to the entire protein sample. Add PEI slowly, with stirring, until the target PEI % is reached. Stir for an additional ten minutes.
Clarify by centrifugation at 10000g for 10min.
Decant supernatant. The lysate can be further purified by affinity chromatography
or, if desired ammonium sulfate precipitation.
Streptomycin sulfate precipitation
(pET CBD Expression System Manual
- NOVAGEN)
Add 50ml 30% (w/v) strept. Sulf.per liter of cell extract. Add the streptomycin sulfate slowly with stirring. After addition, slowly stir the extract at 4°C ON.
Spin the cell extract at 12000g, 4°C, for 30min.
Stir the clarified cell extract for an additional 24 hours at 4°C to complete precipitation.
Once again spin the cell extract at 12000g, 4°C, for 30min to produce
a clarified cell extract.
Polyethyleneimine (PEI) precipitation (Current Protocols in Protein Science, chapter 4)
Between 20 and 80µl of 5% PEI per ml crude containing 4% (dry) nucleic
acids, nearly completely removes nucleic acids. Use pH ~7.5-8.0, where PEI
is completely cationic and nucleic acids are fully anionic.
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