ANALYTICAL
BIOCHEMISTRY
138, 141-143 (1984)
A Method for the Quantitative Recovery of Protein in Dilute Solution
in the Presence of Detergents and Lipids
D. WESSEL
Lehrstuhl
fir
Biochemie
der PJanze,
Untere
AND U. I. FL~GGE
Karsptile
2, 3400
Giittingen,
Federal
Republic
of Germany
Received August 22, 1983
A rapid method based on a defined methanol-chloroform-water
mixture for the quantitative
precipitation of soluble as well as hydrophobic proteins from dilute solutions (e.g., column
chromatography effluents) has been developed. The effectiveness of this method is not affected
by the presence of detergents, lipids, salt, buffers, and @-mercaptoethanol.
KEY WORDS: protein precipitation; TX- 100; lipids; @mercaptoethanol; sodium dodecyl sulfatepolyacrylamide gel electrophoresis.
The quantitative (e.g., by Lowry’s assay) or
the qualitative (e.g., by SDS-PAGE)’ determination of dilute samples of protein containing salt, detergents like TX-100, phospholipids, or P-mercaptoethanol is difficult
because of the interference of these substances
with the analysis methods. Several procedures
which circumvent these problems by initial
removal of the detergent (l-3) or /3-mercaptoethanol (4) prior to the analysis step have
been published. However, these methods are
suitable only if the sample contains enough
protein to be analyzed without further concentration. Dilute protein samples, however,
have to be concentrated. Concentration together with the removal of interfering substances could be attempted by acid precipitation (5-7) and/or precipitation by acetone
or methanol (8). However, it is often difficult
to find optimal precipitation conditions especially when working with hydrophobic (membrane) proteins in the presence of detergents
and/or phospholipids.
In this paper we report a method for the
quantitative precipitation of soluble as well as
i Abbreviations used SDS-PAGE, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis; BSA, bovine
serum albumin; TX-100, Triton X-100; &ME, &mercaptoethanol.
hydrophobic membrane proteins which is not
sensitive to interference by the presence of
salt, detergents, /3-mercaptoethanol, or phospholipids. Samples ready for protein assay or
SDS-gel electrophoresis can be prepared in
less than 20 min.
MATERIALS
AND
METHODS
Bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), TX-100, and asolectin
were purchased from Sigma; methanol and
chloroform from Merck, Darmstadt, FRG;
and rabbit serum from Calbiochem. Membrane proteins from spinach envelopes were
prepared according to Deuce et al. (9). Protein
was analyzed by the method of Lowry ( 10)
and SDS-gel electrophoresis was performed
as described by Laemmli (1 l), using 12.5%
acrylamide and 0.1% NJ’-methylenebisacrylamide.
Procedure. An aliquot (0.4 ml) of methanol
is added to 0.1 ml of protein sample and the
samples are vortexed and centrifuged ( 10 s at
90008) for total collection of the sample. Then
chloroform (0.1 ml) is added and the samples
are vortexed and centrifuged again ( 10 s at
9000g). For samples containing a high concentration of phospholipids (e.g., liposomes)
0.2 ml of chloroform is used. For phase separation 0.3 ml of water is added, and the sam141
0003-2697184 $3.00
Copyright Q 1984 by Academic Press, Inc.
All rights of reproduction in any form reserved.
142
WESSEL AND
pies are vortexed vigorously and centrifuged
for 1 min at 9000g. The upper phase is carefully removed and discarded. A further 0.3
ml methanol is added to the rest of the lower
chloroform phase and the interphase with the
precipitated protein. The samples are mixed
and centrifuged again for 2 min at 9000g to
pellet the protein. The supematant is removed
and the protein pellet is dried under a stream
of air. The dried pellet can be stored now until
use. For protein determination the pellets are
solubilized by the addition of 50-100 ~1 of
SDS (5%) and assayed according to the Lowry
procedure ( 10). For SDS-gel electrophoresis
the pellets are dissolved in 50 ~1 electrophoresis
sample buffer (11) containing 5% SDS.
The above procedure can be reliably
adapted to a larger sample volume. Large
samples (1 ml) are precipitated with 4 ml
methanol, 1 ml chloroform, and 3 ml water.
After centrifugation and removal of the upper
phase the lower phase and the interphase are
transferred to 1.5-ml tubes and the subsequent
procedures are as described above.
RESULTS
OF PROTEIN
PRECIPITATED
AND
DISCUSSION
The above procedure for the quantitative
protein precipitation from solutions containing salt, /3-mercaptoethanol,
or detergents
starts with the addition of 4 parts methanol
and 1 part chloroform to 1 part of sample. A
phase separation is achieved by the addition
of 3 parts water whereby the protein is precipitated at the chloroform-methanol-water
interphase. The addition of an excess of methanol and the subsequent centrifugation results
in a protein pellet which is totally free of interfering substances. Table 1 shows the efficiency of this method for the precipitation of
soluble proteins, e.g., for BSA or for the serum
proteins from rabbit, as well as for hydrophobic membrane proteins (e.g., the proteins
of the chloroplast envelope membrane or
membrane proteins from rat liver). The presence of the anionic detergent SDS (up to 5%)
does not appear to affect the recovery of protein obtained by this method. The same result
is obtained in the presence of the nonionic
TABLE
RECOVERY
FLijGGE
1
IN THE PRESENCE OF OTHER
SUBSTANCES
Amount of protein recovered
Soluble proteins
Membrane proteins from
BSA
Control, not precipitated (pg)
Protein precipitated (yield, %) in
samples containing
5% SDS
1% TX-100
2% TX-100
1% TX-100 plus 20 mg/ml asolectin
1% TX-100 plus 0.5 M KCl
1% TX-100 plus I% /Y-ME
Serum
proteins
(rabbit)
Chloroplast
envelopes
60.9”
40
120
50.0
96 f 1.2
92 + 2.5
100 + 2.3
98 + 1.8
n.d.
95 + 2.1
95 + 4.0
97 + 2.3
92 f 1.8
92 + 4.6
96 -t 3.5
n.d.
100 + 3.8
98 rt 2.2
100 _+ 1.8
94 + 1.5
95 -t 2.3
95 f 2.1
94 + 1.9
n.d.
96 k 2.2
94 +
100b +
100b f
94b +
95b +
98b +
Mitochondria
(rat liver)
76.0“
0.8
0.6
0.6
0.6
2.5
2.2
94 iT 1.5
95b + 2.3
95b + 2.1
94b + 1.9
n.d.
96b f 2.5
Note. The volume was always 100 ~1 containing the given amounts of protein.
0 Membrane proteins were first solubilized in 5% TX- 100 and then diluted to 1% TX- 100. For protein determination
of the not precipitated membrane proteins solubilized in TX-100, SDS was present during the Lowry procedure as
described in ( 12).
b Membrane protein was solubilized in 5% TX-100 and afterward adjusted to the stated conditions.
PROTEIN
RECOVERY
IN DILUTE
SOLUTION
143
ited only by the sensitivity of the protein determination method applied subsequently. So
far, little is known about the molecular weight
cutoffs for this procedure, but even with carbonic anhydrase (M, 30,000), trypsinogen (M,
24,000) and &lactoglobulin (M, 18,400) the
protein recovery is comparable to that outlined
in Table 1 (results not shown). The method
is also effective for the removal of lipids from
membranes with a high lipid/protein ratio.
Due to the high lipid content, a distortion of
the SDS-gel pattern of those membranes, for
example, the outer envelope membrane of
spinach chloroplasts with a lipid/protein ratio
of 3 (13) is observed (Fig. 1, lane 2). Removal
of the lipids by the above method leads to an
improved protein pattern without any apparent quantitative loss of protein (Fig. 1,
lane 3).
ACKNOWLEDGMENT
This research was supported by the Deutsche Forschungsgemeinschaft. (Fl 126/2-2)
FIG. 1. SDS-PAGE of spinach envelope membranes
equivalent to 40-60 rg protein. Lanes 1 and 4, total envelope membrane; 3, outer envelope membranes; 4, outer
envelope membranes precipitated before SDS-PAGE as
described under Materials and Methods.
detergent TX- 100, which is a common reagent
in membrane biochemistry. Quantitative recovery of protein is also obtained when the
samples in addition to TX- 100 also contain
P-mercaptoethanol (up to 1%) or salts like potassium chloride. These substances are quantitatively removed during the precipitation
procedure, so that this simple method is applicable for total recovery of both soluble and
hydrophobic membrane proteins. As pointed
out already, this method avoids heating or
acid conditions for the precipitation of protein.
Therefore this procedure is also useful for the
removal of protein from extracts which are to
be analyzed for various substrates labile to
extreme pH or heat.
Quantitative recovery of protein can be obtained in even more dilute protein solutions,
and 2-3 pg protein can be quantitatively precipitated so that the method seems to be lim-
REFERENCES
1. Mather, J. H., and Tamplin, C. B. (1979) Anal.
Biochem. 93, 139- 142.
2. ChandraRajan, J., and Klein, L. (1975) Anal. B&hem.
69, 632-636.
3. Horikawa, S., and Ogawara, H. (1979) Anal. B&hem.
97, 116-I 19.
4. Makkar, H. P. S., Sharma, 0. P., and Negi, S. S.
(1980) Anal. B&hem. 104, 124-126.
5. Schaffner, W., and Weissmann, C. (1973) Anal.
Biochem. 56, 502-5 14.
6. Retz, K. C., and Steele, W. J. (1977) Anal. Biochem.
79,457-461.
7. Bensadoun, A., and Weinstein, D. (1976) Anal.
Biochem. 70, 24 l-250.
8. Hudgin, R. L., Priecer, W. E., and Ashwell, G. (1974)
J. Biol. Chem. 249, 5536-5543.
9. Deuce, R., Holtz, R. B., and Benson, A. A. (1973)
J. Biol. Chem. U&7215-7222.
10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and
Randall, R. J. (1957) J. Biol. Chem. 193, 265275.
11. Laemrali, U. K. (1970) Nature (London) 227, 680685.
12. Wang, C. S., and Smith, R. L. (1975) Anal. Biochem.
63,414-417.
13. Dome, J., Block, H. A., Joyard, J., and Deuce, R.
(1982) in Biochemistry and Metabolism of Plant
Lipids (Wintermans, J. F. G. M., and Kuiper,
R. J. C., eds.), pp. 153-164, Elsevier Biomedical
Press, Amsterdam.