CHAPTER SEVEN
Salting out of Proteins Using
Ammonium Sulfate Precipitation
Krisna C. Duong-Ly, Sandra B. Gabelli1
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
1
Corresponding author: e-mail address: gabelli@jhmi.edu
Contents
1. Theory
2. Equipment
3. Materials
3.1 Solutions & buffers
4. Protocol
4.1 Preparation
4.2 Duration
5. Step 1 Removal of Proteins Marginally Soluble in (NH4)2SO4
5.1 Overview
5.2 Duration
5.3 Tip
6. Step 2 Precipitation of the Protein of Interest
6.1 Overview
6.2 Duration
6.3 Tip
6.4 Tip
6.5 Tip
6.6 Tip
6.7 Tip
References
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Abstract
Protein solubility is affected by ions. At low ion concentrations (<0.5 M), protein solubility increases along with ionic strength. Ions in the solution shield protein molecules from
the charge of other protein molecules in what is known as ‘salting-in’ (Fig. 7.1). At a very
high ionic strength, protein solubility decreases as ionic strength increases in the process
known as ‘salting-out’. Thus, salting out can be used to separate proteins based on their
solubility in the presence of a high concentration of salt. In this protocol, ammonium
sulfate will be added incrementally to an E. coli cell lysate to isolate a recombinantly
over-expressed protein of 20 kDa containing no cysteine residues or tags.
Methods in Enzymology, Volume 541
ISSN 0076-6879
http://dx.doi.org/10.1016/B978-0-12-420119-4.00007-0
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2014 Elsevier Inc.
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85
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Krisna C. Duong-Ly and Sandra B. Gabelli
Figure 7.1 (a) Dependence of solubility on salt concentration. Salting-in increases protein solubility at low salt concentrations. As salt concentration is increased further, proteins are salted-out (Zhou, 2005). (b) Solubility of horse carboxyhemoglobin in the
presence of sodium chloride, potassium chloride, magnesium sulfate and ammonium
sulfate. Curves are idealized from data (Green and Hughes, 1955).
1. THEORY
Few proteins are soluble only in water and most require at least a small
concentration of salt to remain folded and stable. Often, proteins that contain positively and negatively charged regions self-aggregate under very low
salt conditions. When salt is present, however, the anions and cations neutralize charges on the protein surface, preventing aggregation. As the salt
concentration is increased even further, the surface of the protein will
become so charged that once again, the protein molecules will aggregate
(Green and Hughes, 1955; Scopes, 1993). The salting-out ability of multiply
charged anions such as sulfate follows the Hofmeister series.
Hofmeister series for anions:
PO4 3 > SO4 2 > CH3 COO > Cl > Br > ClO4 > I > SCN
Hofmeister series for cations:
NH4 þ > Rbþ > Kþ > Naþ > Liþ > Mg2þ > Ca2þ > Ba2þ
Ammonium sulfate, (NH4)2SO4, is often used for salting out because of
its high solubility, which allows for solutions of very high ionic strength, low
price, and availability of pure material. Additionally, NH4þ and SO42 are at
the ends of their respective Hofmeister series and have been shown to stabilize protein structure (Burgess, 2009). Some proteins follow a reversal of
Salting out of Proteins Using Ammonium Sulfate Precipitation
87
the Hofmeister effect when pH <pI (e.g., lysozyme and crystallins)
(Bostrom et al., 2005).
Salting out removes proteins that easily aggregate from those that are very
soluble making it a good initial purification step for small soluble proteins
(Englard and Seifter, 1990). It is one of the few protein purification methods
that does not require the presence of a purification tag on the protein (see also
Using ion exchange chromatography to purify a recombinantly expressed
protein, Gel filtration chromatography (Size exclusion chromatography) of
proteins, Use and Application of Hydrophobic Interaction Chromatography
for Protein Purification and Hydroxyapatite Chromatography: Purification
Strategies for Recombinant Proteins); thus, if the protein is naturally expressed
in the host cell, cloned in the absence of tags, or has an unexposed tag, salting
out is an ideal choice for purification. This method may also be applied to
purify a protein of unknown sequence. Since the sample is in a high concentration of (NH4)2SO4 at the end of the experiment, hydrophobic interaction
chromatography (HIC) may be used immediately to further purify the sample.
The primary shortcoming of using salting out to purify proteins, however, is that contaminants often precipitate with the protein of interest. To
obtain a pure protein sample, further purification steps such as ion exchange
chromatography (see Using ion exchange chromatography to purify a
recombinantly expressed protein) and gel filtration chromatography (see
Gel filtration chromatography (Size exclusion chromatography) of proteins)
are needed. Also, the protein is in a high concentration of salt at the end of
the experiment. Dialysis is generally the best method to remove (NH4)2SO4
from a sample. Applying a sample with a high concentration of (NH4)2SO4
to a desalting column is unadvisable as this may cause the column medium to
compress.
2. EQUIPMENT
Refrigerated high-speed centrifuge
Magnetic stir plate
Polyacrylamide gel electrophoresis equipment
Oak Ridge polycarbonate centrifuge tubes
Beakers
Graduated cylinders
Magnetic stir bar
1.5-ml microcentrifuge tubes
Micropipettors
Micropipettor tips
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Krisna C. Duong-Ly and Sandra B. Gabelli
3. MATERIALS
Ammonium sulfate [(NH4)2SO4]
Tris-hydrochloride (Tris-HCl)
Dithiothreitol (DTT)
Materials for SDS-PAGE
Coomassie Brilliant Blue R250 stain
3.1. Solutions & buffers
Step 1 Lysis buffer
Component
Final concentration
Stock
Amount
Tris–HCl, pH 7.5
50 mM
1M
50 ml
DTT
0.1 mM
1M
0.1 ml
Add water to 1 l
4. PROTOCOL
4.1. Preparation
Lyse the cells in the lysis buffer. Harvest the cell lysate through centrifugation
and save the pellet for later analysis by SDS-PAGE.
4.2. Duration
Preparation
About 2 h
Protocol
About 5–6 h
See Fig. 7.2 for the flowchart of the complete protocol.
Figure 7.2 Flowchart of the complete protocol, including preparation.
Salting out of Proteins Using Ammonium Sulfate Precipitation
89
5. STEP 1 REMOVAL OF PROTEINS MARGINALLY
SOLUBLE IN (NH4)2SO4
5.1. Overview
Add a small amount of (NH4)2SO4 to the cell lysate to remove any proteins
easily precipitated by (NH4)2SO4.
5.2. Duration
1 h 15 min
1.1 Measure the volume of the cell lysate. Refer to Table 7.1 to determine
the amount of (NH4)2SO4 to add to bring the lysate to 30% saturation
(Dawson et al., 1969).
1.2 Pour the cell lysate into a centrifuge bottle or large beaker and add the
appropriate amount of (NH4)2SO4. Add the stir bar and stir in the cold
room for 30 min.
1.3 Remove the stir bar and collect the precipitated proteins by centrifugation at 20 000 g for 30 min.
1.4 Remove the supernatant and save. Reserve an aliquot for analysis
by SDS-PAGE. Resuspend the pellet in a minimal volume of
lysis buffer and store on ice or at 4 C for later analysis by
SDS-PAGE.
5.3. Tip
In this protocol, 30% saturation is meant only as an initial guideline. If the protein of
interest precipates at this concentration, decrease this amount to 10% or 15%. This
step simply serves to remove large aggregates that may not have been removed during
the harvesting of the cell lysate during the preparation step.
See Fig. 7.3 for the flowchart of Step 1.
6. STEP 2 PRECIPITATION OF THE PROTEIN OF INTEREST
6.1. Overview
Increase the saturation of the cell lysate and precipitate the desired
protein.
Table 7.1 Final concentration of ammonium sulfate: percentage saturation at 0 Ca
Percentage saturation at 0 C
20
Initial concentration of
ammonium sulfate
(percentage saturation at 0 C)
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Solid ammonium sulfate (grams) to be added to
100 ml of solution
0
10.6 13.4 16.4 19.4 22.6 25.8 29.1 32.6 36.1 39.8 43.6 47.6 51.6 55.9 60.3 65.0 69.7
5
7.9 10.8 13.7 16.6 19.7 22.9 26.2 29.6 33.1 36.8 40.5 44.4 48.4 52.6 57.0 61.5 66.2
10
5.3
8.1 10.9 13.9 16.9 20.0 23.3 26.6 30.1 33.7 37.4 41.2 45.2 49.3 53.6 58.1 62.7
15
2.6
5.4
8.2 11.1 14.1 17.2 20.4 23.7 27.1 30.6 34.3 38.1 42.0 46.0 50.3 54.7 59.2
20
0
2.7
5.5
8.3 11.3 14.3 17.5 20.7 24.1 27.6 31.2 349 38.7 42.7 46.9 51.2 55.7
0
2.7
5.6
8.4 11.5 14.6 17.6 21.1 24.5 28.0 31.7 35.5 39.5 43.6 47.8 52.2
0
2.8
5.6
8.6 11.7 14.8 18.1 21.4 24.9 28.5 32.3 36.2 40.2 44.5 48.8
0
2.8
5.7
8.7 11.8 15.1 18.4 21.8 25.4 29.1 32.9 36.9 41.0 45.3
0
2.9
5.8
8.9 12.0 15.3 18.7 22.2 25.8 29.6 33.5 37.6 41.8
0
2.9
5.9
25
30
35
40
45
9.0 12.3 15.6 19.0 22.6 26.3 30.2 34.2 38.3
50
55
60
65
70
75
80
85
90
95
0
3.0
6.0
9.2 12.5 15.9 19.4 23.0 26.3 30.8 34.8
0
3.0
6.1
9.3 12.7 16.1 19.7 23.5 27.3 31.3
0
3.1
6.2
9.5 12.9 16.4 20.1 23.9 27.6
0
3.1
6.3
9.7 13.2 16.8 20.5 24.4
0
3.2
6.5
9.9 13.4 17.1 20.9
0
3.2
6.6 10.1 13.7 17.4
0
3.3
0
6.7 10.3 13.9
3.4
0
6.8 10.5
3.4
7.0
0
3.5
100
a
Adapted from Dawson RMC, Elliott DC, Elliott WH, and Jones KM (eds.) (1969) Data for Biochemical Research, 2nd edn. London: Oxford University Press.
0
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Krisna C. Duong-Ly and Sandra B. Gabelli
Figure 7.3 Flowchart of Step 1.
6.2. Duration
About 3–4 h
2.1 Measure the volume of the supernatant from Step 1. Refer to Table 7.1
to determine the amount of (NH4)2SO4 to add to bring the lysate to
60% saturation.
2.2 Pour the cell lysate into a centrifuge bottle or large beaker and add the
appropriate amount of (NH4)2SO4. Add the stir bar and stir in the cold
room for 30 min.
2.3 Remove the stir bar and remove the precipitated proteins by centrifugation at 20 000 g for 30 min.
2.4 Remove the supernatant and save. Reserve an aliquot for analysis by
SDS-PAGE. Resuspend the pellet in a minimal volume of lysis buffer
and store on ice or at 4 C for later analysis by SDS-PAGE.
2.5 Measure the volume of the supernatant from Step 2.4. Refer to
Table 7.1 to determine the amount of (NH4)2SO4 to add to bring
the lysate to 90% saturation.
2.6 Pour the cell lysate into a centrifuge bottle or large beaker and add the
appropriate amount of (NH4)2SO4. Add the stir bar and stir in the cold
room for 30 min.
2.7 Remove the stir bar and remove the precipitated proteins by centrifugation at 20 000 g for 30 min.
2.8 Remove the supernatant and save. Reserve an aliquot for analysis by
SDS-PAGE. Resuspend the pellet in a minimal volume of lysis buffer
and store on ice or at 4 C for later analysis by SDS-PAGE.
Salting out of Proteins Using Ammonium Sulfate Precipitation
93
2.9 Determine where the protein of interest is found using SDS-PAGE (see
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDSPAGE)). Be sure to include samples of the E. coli cell lysate and of each
pellet and supernatant after each round of precipitation. Stain gel with
Coomassie blue to visualize proteins (see Coomassie Blue Staining).
6.3. Tip
The concentrations of (NH4)2SO4 used in this step can be modified to achieve optimal
separation. Also, increasing the (NH4)2SO4 saturation can be achieved in more than
a total of three steps to improve separation.
6.4. Tip
Generally, few proteins do not precipate in a solution of 90% (NH4)2SO4 saturation. However, if after the 90% step your protein is still in the supernatant, save this
sample but do not try to increase saturation further as this is approaching the limit of
solubility for (NH4)2SO4.
6.5. Tip
On SDS-PAGE gels, the samples with high (NH4)2SO4 may run anomalously
because of the high salt content. Consider diluting the samples with a buffer containing
no salt.
6.6. Tip
The purified protein in ammonium sulfate may be filtered and then applied to an HIC
column or saved at 4 C. The presence of the high ionic strength buffer generally
stabilizes proteins.
6.7. Tip
Even for pure samples, a precipitation with (NH4)2SO4 is a great way to concentrate
large volumes of protein (i.e., add 90% (NH4)2SO4, stir, centrifuge and redissolve
the pellet in the smallest possible amount of buffer). This will, however, increase the
concentration of salt in the sample.
See Fig. 7.4 for the flowchart of Step 2.
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Krisna C. Duong-Ly and Sandra B. Gabelli
Figure 7.4 Flowchart of Step 2.
REFERENCES
Referenced Literature
Green, A., & Hughes, W. (1955). Methods in Enzymology, 1, 67.
Scopes, R. (1993). In C. R. Cantor (Ed.), Protein Purification: Principles and Practice
(pp. 71–101) (3rd ed.) New York: Springer.
Burgess, R. R. (2009). Methods in Enzymology, 463, 331.
Bostrom, M., et al. (2005). Biophysical Chemistry, 117, 217.
Englard, S., & Seifter, S. (1990). Methods in Enzymology, 182, 285.
Dawson, R. M. C., Elliott, D. C., Elliot, W. H., & Jones, K. M. (Eds.), (1969). Data for
Biochemical Research. London: Oxford University Press.
Zhou, H. X. (2005). Proteins, 61, 69.
Referenced Protocols in Methods Navigator
Using ion exchange chromatography to purify a recombinantly expressed protein.
Gel filtration chromatography (Size exclusion chromatography) of proteins.
Use and Application of Hydrophobic Interaction Chromatography for Protein Purification.
Hydroxyapatite Chromatography: Purification Strategies for Recombinant Proteins.
One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE).
Coomassie Blue Staining.