Affinity Chromatography - Department of Molecular and Cellular ...

Examples of precipitation agents are reviewed in Table 13. The most common precipitation
method using ammonium sulphate is described in more detail.
Table 13. Examples of precipitation techniques.
Precipitation agent Typical conditions for use Sample type Comment
Ammonium sulphate As described below. > 1 mg/ml proteins Stabilizes proteins, no
especially immuno- denaturation, supernatant
globulins.
can go directly to HIC.
Dextran sulphate Add 0.04 ml 10% Samples with high levels Precipitates lipoprotein.
dextran sulphate and of lipoprotein e.g ascites.
1 ml 1 M CaCl 2 per
ml sample, mix 15 min,
centrifuge 10 000 g,
discard pellet.
Polyvinylpyrrolidine Add 3% (w/v), stir 4 hours, Samples with high levels Alternative to dextran
centrifuge 17 000 g, of lipoprotein e.g ascites. sulphate.
discard pellet.
Polyethylene glycol Up to 20% w/vol Plasma proteins. No denaturation,
(PEG, M r > 4000)
supernatant goes directly
to IEX or AC, complete
removal may be difficult.
Acetone (cold) Up to 80% vol/vol at +0 °C. May denature protein
Collect pellet after
irreversibly.
centrifugation at full speed
Useful for peptide
in an Eppendorf centrifuge.
precipitation or concentration
of sample for electrophoresis.
Polyethyleneimine 0.1% w/v Precipitates aggregated
nucleoproteins.
Protamine sulphate 1% w/v Precipitates aggregated
nucleoproteins.
Streptomycin sulphate 1% w/v Precipitation of nucleic acids.
Caprylic acid (X/15) g where X = volume Antibody concentration Precipitates bulk of proteins
of sample. should be > 1 mg/ml. from sera or ascites, leaving
immunoglobulins in solution.
Details taken from:
Scopes R.K., Protein Purification, Principles and Practice, Springer, (1994), J.C. Janson and L. Rydén, Protein
Purification, Principles, High Resolution Methods and Applications, 2nd ed. Wiley Inc, (1998).
Personal communications.
Ammonium sulphate precipitation
Some proteins may be damaged by ammonium sulphate. Take care when adding crystalline
ammonium sulphate: high local concentrations may cause contamination of the precipitate
with unwanted proteins.
For routine, reproducible purification, precipitation with ammonium sulphate should be
avoided in favour of chromatography.
In general, precipitation is rarely effective for protein concentrations below 1 mg/ml.
Solutions needed for precipitation:
Saturated ammonium sulphate solution (add 100 g ammonium sulphate to 100 ml distilled water, stir to dissolve).
1 M Tris-HCl, pH 8.0.
Buffer for first purification step.
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