Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.

prot4308

Protocol

Bulk Precipitation of Proteins by Ammonium Sulfate

Richard J. Simpson

This protocol was adapted from "Concentrating Solutions of Protein," in Appendix 3 of Purifying Proteins
for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.

INTRODUCTION

This protocol can be used to concentrate bulk proteins from large volumes of solution.
Few proteins precipitate when the concentration of ammonium sulfate in the solution is
<25% saturation, whereas a concentration of ~75% saturation at 4ºC is high enough to
precipitate most proteins. Higher concentrations of ammonium sulfate (up to 100%
saturation) increase the density of the protein suspension, making it more difficult to
recover the protein by centrifugation.

MATERIALS

Reagents

Ammonium sulfate (analar grade)

Resuspension buffer

This buffer is used to resuspend the concentrated protein after ammonium sulfate
precipitation. The choice of buffer will depend on the subsequent application.

Solution containing the target protein

Since ammonium sulfate solutions are acidic, it is important to buffer the protein solution
at a neutral pH with an appropriate buffer (e.g., 50 mM Tris-Cl at pH 7.2). If the target
protein is inactivated or inhibited by metals, the buffer should contain a chelating agent
such as EDTA (10 mM) to remove heavy-metal cations present even in analar grade
ammonium sulfate. To ensure optimal precipitation, it is best to work with solutions
containing >1 mg protein/ml.

Equipment
Glass rod

Pestle and mortar

Screw-top polycarbonate centrifuge tubes or bottles

METHOD

1. Use a graduated cylinder to measure the volume of the protein solution to be

concentrated. Pour the solution into a beaker with a capacity approximately twice
the measured volume of the protein solution. Place the beaker in a bed of ice.

2. Carry out either Step 2.i or 2.ii.

i. In the ammonium sulfate tables (Table 1 and Table 2), find the amount of solid
ammonium sulfate required to give the desired percentage of saturation. Weigh out
the necessary amount of ammonium sulfate. If the ammonium sulfate contains
lumps, use a mortar and pestle to crush the lumps to a smooth powder.

or

ii. Determine the volume of saturated ammonium sulfate solution (see Table 1 and
Table 3) necessary to give the desired percentage of saturation. Pour the required
amount of ammonium sulfate solution into a graduated cylinder.
3. Place the beaker containing the chilled protein solution (and a large magnetic
stir bar) on a large stir plate in the cold room.

4. Sprinkle small batches of the solid ammonium sulfate onto the surface of the
stirring protein solution (or pour small amounts of the saturated ammonium sulfate
solution) so that it takes ~30 minutes to add all of the ammonium sulfate. Use a
slow rate of stirring to avoid foaming. If solid ammonium sulfate accumulates at
the bottom of the beaker, stop the addition and allow the ammonium sulfate to
dissolve before adding more solid.

IMPORTANT: The stirring must be as gentle and even as possible, otherwise

protein denaturation will occur (as evidenced by foaming). Avoid using a
magnetic stirrer that generates a significant amount of heat during stirring.

5. After the addition of ammonium sulfate is complete, stir the mixture for another
60 minutes to ensure complete precipitation.

Most proteins precipitate within 15-20 minutes after the final addition of
ammonium sulfate, but others need several hours to precipitate completely.

6. Transfer the mixture to screw-cap polycarbonate centrifuge tubes or bottles, and

carefully balance them. Ensure that the centrifuge tubes are balanced to within 0.1-
0.2 g across the rotor axis.
 7. Centrifuge the tubes at 10,000g for 15 minutes (or at 5000g for 30 minutes) at
4°C.

Since ammonium sulfate is very corrosive, wash the centrifuge rotors in warm
water soon after use.

8. Decant the supernatant from each centrifuge tube.

9. Resuspend the protein pellet in the minimum volume (typically 1-2 pellet
volumes) of resuspension buffer, as follows:
i. Add a few milliliters of buffer to one of the pellets, and stir with a glass rod to
resuspend the protein.

ii. If several centrifuge tubes have been used, transfer the protein suspension to a
second tube containing a pellet, and stir with a glass rod to resuspend the protein.

iii. Repeat Step ii until all of the pellets have been resuspended. Add a few more
milliliters of buffer (or H2O) whenever the suspension becomes too thick to
transfer.

iv. After the final transfer, use a small volume of buffer to rinse out the centrifuge
tubes, and add the washings to the final suspension.

Proteins are generally stable in ammonium sulfate and can be safely stored as an
ammonium sulfate suspension for several days at 4°C at this stage of a
purification procedure.

10. If necessary, centrifuge the resultant protein solution at 10,000g for 15 minutes
to remove any remaining insoluble material (e.g., denatured protein).

11. If necessary, remove any residual ammonium sulfate from the protein solution
by dialysis, ultrafiltration, or chromatographic desalting.

Removal of ammonium sulfate is necessary if the protein is next going to be

subjected to ion-exchange chromatography. However, the presence of ammonium
sulfate will not interfere with protein separation by hydrophobic or size-exclusion
chromatography.

12. If desired, assay the protein solution for the presence of the target protein by
SDS-PAGE and/or rapid western blot analysis, and for total protein.

REFERENCES

Dawson R.M.C., Elliott D.C., Elliott W.H., and Jones K.M., eds. 1986. Data for
biochemical research, 3rd ed. Clarendon Press, Oxford, United Kingdom.
Scopes R.K. 1987. Protein purification: Principles and practice, 2nd ed. Springer-Verlag,
New York.

Taylor J.F. 1953. The isolation of proteins. In The proteins: Chemistry, biological activity,
and methods, part A (eds. H. Neurath and K.C. Bailey), pp. 2-72. Academic Press, New
York.

Caution

Ammonium sulfate
Ammonium sulfate, (NH4)2SO4, may be harmful by inhalation, ingestion, or skin
absorption. Wear appropriate gloves and safety glasses.

Table

Table 1. Saturated solutions of ammonium sulfate in

water at several temperatures

Temperature Grams Grams Moles Percentage Molarity of Apparent VG/1000c

(°C) (NH4)2SO4 (NH4)2SO4 (NH4)2SO4 by weight saturated specific
required to added per in 1000 g of (NH4)2SO4 volume in
saturate liter of H2O solution saturated
1000 g of saturated solutionc
H2Oa,b solutionc

0 706.86 514.72 5.35 41.42 3.90 0.5262 0.271

10 730.53 525.05 5.53 42.22 3.97 0.5357 0.281
20 755.82 536.34 5.73 43.09 4.06 0.5414 0.290
25 766.80 541.24 5.82 43.47 4.10 0.5435 0.294
30 777.55 545.88 5.91 43.85 4.13 0.5458 0.298

Adapted from Dawson et al. (1986) and Taylor (1953).

a
Note that the volume changes upon addition of solid (NH4)2SO4.
b
The degree of saturation is usually considered to be that which is calculated for the
addition of a saturated solution without allowing for a volume change upon mixing. For
example, 1 volume of water plus 1 volume of saturated (NH4)2SO4 is considered to yield a
solution that is 0.5 (50%) saturated. According to this convention, the volume (V) of
saturated (NH4)2SO4, which has to be added to 100 ml of solution of initial saturation S1 to
produce a final saturation S2 is given by the equation: V = 100(S2 - S1)/(1 - S2), where S1
and S2 are expressed as fractions of the saturated solution. For example, if S1 = 0.5 and S2
= 0.7, then V = 66.67 ml.
c
When preparing more concentrated solutions of (NH4)2SO4, it is more economical and
often more convenient to add the solid salt instead of a stock solution of saturated
(NH4)2SO4 (see Table 2). The weight in grams, X, to be added to 100 ml of solution of
saturation S1 to yield a solution of saturation S2 may be calculated as follows: X = 0.1G(S2
- S1)/(1 - (VG/1000)S2), where G = grams of (NH4)2SO4 in 1000 ml of saturated solution,
and V = apparent specific volume of (NH4)2SO4 in a saturated solution. Values of G, V,
and VG/1000 at different temperatures are given in columns 3, 7, and 8, respectively. For
example, if S1 = 0.5 and S2 = 0.7, at 0°C, G = 514.72 and VG/1000 = 0.271, then X = 20.2
g.

Table

Table 2. Ammonium sulfate saturation table using solid

ammonium sulfate

Initial Final concentration of ammonium sulfate(% saturation at 0°C)

concentration
of ammonium
sulfate(%
saturation at
0°C)

20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
Grams solid ammonium sulfate to add to 100 ml of solution
0 10.7 13.6 16.6 19.7 22.9 26.2 29.5 33.1 36.6 40.4 44.2 48.3 52.3 56.7 61.1 65.9
5 8.0 10.9 13.9 16.8 20.0 23.2 26.6 30.0 33.6 37.3 41.1 45.0 49.1 53.3 57.8 62.4
10 5.4 8.2 11.1 14.1 17.1 20.3 23.6 27.0 30.5 34.2 37.9 41.8 45.8 50.0 54.4 58.9
15 2.6 5.5 8.3 11.3 14.3 17.4 20.7 24.0 27.5 31.0 34.8 38.6 42.6 46.6 51.0 55.5
20 0 2.7 5.6 8.4 11.5 14.5 17.7 21.0 24.4 28.0 31.6 35.4 39.2 43.3 47.6 51.9

25 0 2.7 5.7 8.5 11.7 14.8 18.2 21.4 24.8 28.4 32.1 36.0 40.1 44.2 48.5
30 0 2.8 5.7 8.7 11.9 15.0 18.4 21.7 25.3 28.9 32.8 36.7 40.8 45.1
35 0 2.8 5.8 8.8 12.0 15.3 18.7 22.1 25.8 29.5 33.4 37.4 41.6
40 0 2.9 5.9 9.0 12.2 15.5 19.0 22.5 26.2 30.0 34.0 38.1
45 0 2.9 6.0 9.1 12.5 15.8 19.3 22.9 26.7 30.6 34.7

50 0 3.0 6.1 9.3 12.7 16.1 19.7 23.3 27.2 31.2

55 0 3.0 6.2 9.4 12.9 16.3 20.0 23.8 27.7
60 0 3.1 6.3 9.6 13.1 16.6 20.4 24.2
65 0 3.1 6.4 9.8 13.4 17.0 20.8
70 0 3.2 6.6 10.0 13.6 17.3

75 0 3.2 6.7 10.2 13.9

80 0 3.3 6.8 10.4
85 0 3.4 6.9
90 0 3.4
95 0
100

Adapted from Dawson et al. (1986).

Values given are the amount of solid (NH4)2SO4 required to bring a solution of known
initial saturation to a desired final saturation. For the data listed, the saturation is relative
to full saturation at 0°C.

Table

Table 3. Ammonium sulfate saturation table using

saturated ammonium sulfate solution

Initial Final concentration of ammonium sulfate(% v/v)

concentration
of ammonium
sulfate(% v/v)

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
Milliliters saturated ammonium sulfate solution to add to 100 ml of solutiona,b
0 11 18 25 33 43 54 67 82 100 122 150 186 233 300 400 567 900 1900
5 6 12 19 26 36 46 58 73 90 111 138 171 217 280 375 533 850 1800
10 0 6 13 20 29 38 50 64 80 100 125 157 200 260 350 500 800 1700
15 0 6 13 21 31 42 55 70 89 113 143 183 240 325 466 750 1600

20 0 6 14 23 33 45 60 78 100 129 167 220 300 433 700 1500

25 0 7 15 25 36 50 67 88 114 150 200 275 400 650 1400
30 0 8 17 27 40 56 75 100 133 180 250 367 600 1300
35 0 8 18 30 44 63 86 117 160 225 333 550 1200
40 0 9 20 33 50 71 100 140 200 300 500 1100
45 0 10 22 38 57 83 120 175 267 450 1000

50 0 11 25 43 67 100 150 233 400 900

55 0 13 29 50 80 125 200 350 800
60 0 14 33 60 100 167 300 700
65 0 17 40 75 133 250 600
70 0 20 50 100 200 500

75 0 25 67 150 400
80 0 33 100 300
85 0 50 200
90 0 100

Adapted from Scopes (1987).

Values given are the amount of saturated (NH4)2SO4 solution required to bring a solution
of known initial (NH4)2SO4 concentration to a desired final (NH4)2SO4 concentration. This
table works equally well for determining the volume of a miscible solvent (such as
ethanol or acetone) to be used for precipitation of proteins.
a
Note that the volume increases (sometimes significantly) as a result of addition of the
precipitant.
b
The values in this table are based on the following equation: Volume of saturated
(NH4)2SO4 to add to 100 ml = 100(C2 - C1)/(100 - C2), where C1 is the initial concentration
of ammonium sulfate (% v/v) and C2 is the final concentration of ammonium sulfate (%
v/v).

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