Fractional Precipitation Protocol for Affinity Chromatography Samples

Appendix 1 extracted from Affinity Chromatography Principles and Methods GE Healthcare, 2007

Fractional precipitation is frequently used at laboratory scale to remove gross impurities from small sample volumes and occasionally used in small-scale commercial production. Precipitation techniques separate fractions by the principle of differential solubility. Because protein species differ in their degree of hydrophobicity, increased salt concentrations can enhance hydrophobic interactions between the proteins and cause precipitation. Fractional precipitation can be applied to remove gross impurities in three different ways, as shown in Figure 75.

Figure 75. Three ways to use precipitation.

Table 13. The most common precipitation method using ammonium sulphate is described in more detail.

Precipitation agent	Typical conditions for use	Sample type	Comment
Ammonium sulphate	As described below.	1 mg/ml proteins especially immuno-globulins.	Stabilizes proteins, no denaturation, supernatant can go directly to HIC. Helps to reduce lipid content.
Dextran sulphate	Add 0.04 ml 10% dextran sulphate and 1 ml 1 M CaCl2 per ml sample, mix 15 min, centrifuge 10 000 g, discard pellet.	Samples with high levels of lipoprotein e.g ascites.	Precipitates lipoprotein.
Polyvinylpyrrolidine	Add 3% (w/v), stir 4 hours, centrifuge 17 000 g, discard pellet.	Samples with high levels of lipoprotein e.g ascites.	Alternative to dextran sulphate.
Polyethylene glycol (PEG, Mr > 4000)	Up to 20% w/vol	Plasma proteins.	No denaturation, supernatant goes directly to IEX or AC, complete removal may be difficult. Stabilizes proteins.
Acetone (cold)	Up to 80% vol/vol at +0 °C. Collect pellet after centrifugation at full speed in an Eppendorf™ centrifuge.		May denature protein  irreversibly. Useful for peptide 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 of sample.	Antibody concentration should be > 1 mg/ml.	Precipitates bulk of proteins 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 favor 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.
• Filter (0.45 µm) or centrifuge the sample (10 000 g at +4 °C).
• Add 1 part 1 M Tris-HCl, pH 8.0 to 10 parts sample volume to maintain pH.
• Stir gently. Add ammonium sulphate solution, drop by drop. Add up to 50% saturation*. Stir for 1 hour.
• Centrifuge 20 minutes at 10 000 g.
• Remove supernatant. Wash the pellet twice by resuspension in an equal volume of ammonium sulphate solution of the same concentration (i.e. a solution that will not redissolve the precipitated protein or cause  further precipitation). Centrifuge again.
• Dissolve pellet in a small volume of the buffer to be used for the next step.
• Ammonium sulphate is removed during clarification/buffer exchange steps with Sephadex G-25, using desalting columns (see page 133, Buffer exchange and desalting for affinity chromatography).

*The % saturation can be adjusted either to precipitate a target molecule or to precipitate contaminants.

The quantity of ammonium sulphate required to reach a given degree of saturation varies according to temperature. Table 14 shows the quantities required at +20 °C.

Table 14. Quantities of ammonium sulphate required to reach given degrees of saturation at +20 °C.