Solubilization of Trichloroacetic Acid (TCA) Precipitated Microbial
Proteins via NaOH for Two-Dimensional Electrophoresis
M. P. Nandakumar, Jie Shen, Babu Raman, and Mark R. Marten*
Department of Chemical & Biochemical Engineering, University of Maryland, Baltimore County (UMBC),
1000 Hilltop Circle, Baltimore, Maryland 21250
Received June 23, 2002
Abstract: In preparing intracellular microbial samples for
one- or two-dimensional electrophoresis, trichloroacetic
acid (TCA) precipitation is frequently used to remove
interfering compounds. Solubilization of TCA precipitate
typically requires the addition of a number of chaotropes
or detergents, in a multistep process, that requires hours
to carry out. In this study, a simple, rapid, one-step
method to solubilize TCA precipitated proteins is pre-
sented. Precipitated proteins are pretreated with 0.2 M
NaOH for less than 5 min, followed by addition of
standard sample solubilization buffer (SSSB). When com-
pared to solubilization with SSSB alone, NaOH pretreat-
ment of TCA-precipitated intracellular protein from As-
pergillus oryzae and Escherichia coli shows an approximate
5-fold increase in soluble protein. In addition, two-
dimensional gel electrophoresis on resolubilized proteins
shows an equivalent number of proteins in samples with
and without NaOH pretreatment.
Key
s: proteins ¥ proteomics ¥ Aspergillus oryzae ¥ Escheri-
chia coli
Introduction
The preparation of intracellular microbial proteins for two-
dimensional gel electrophoresis (2-DE) typically includes lysis
of the cell membrane or wall, inactivation or removal of
interfering substances, and solubilization of sample proteins.
During this process, it is of great importance to minimize
protein modification or degradation, thus avoiding a quantita-
tive loss of high molecular weight proteins, membrane proteins,
and/or nuclear proteins.1 As such, three important goals in
sample preparation are as follows: (i) complete dissociation
of protein/protein interactions, (ii) removal of nonprotein
sample components (e.g., lipids, nucleic acids, and salts), and
(iii) significant reduction or elimination of protease activity.2
Although the second and third items can be addressed indi-
vidually by dialysis and addition of protease inhibitors, to a
certain extent, all three goals can be met in a single step:
precipitation of sample proteins followed by resuspension in
sample solubilization buffer.3
Precipitation of proteins using trichloroacetic acid (TCA;
alone, or in combination with acetone) is a popular sample
preparation method for both 1-D and 2-D electrophoresis,4,5
because it can concentrate samples, remove salts, and polysac-
charides,6 and denature endogenous proteases.7,8 However,
resolubilization of precipitated protein is often difficult,4,5,8 and
this is especially true for proteins precipitated with TCA.9-11
For example, current protocols for resolubilization of TCA
precipitate are quite time-consuming4 (Table 1), and can take
up to 24 h. Alternatively, this time can be reduced to ap-
proximately 3 to 4 h by addition of a number of solubilizing
agents (e.g., chaotropes, surfactants, reducing agents, etc.).5,8-11
These, however, can lead to protein modifications or aggrega-
tion, which have the potential to interfere with 2-DE.1 A better
method for resolubilization of TCA precipitated protein is
needed.
In resolubilizing protein precipitate for 1-D gels12 or total
protein determination,13 pretreatment with alkali solution has
been used to enhance solubility. In this study, we report on a
modified alkali pretreatment method for resolubilization of TCA
precipitated proteins for 2-DE (Table 1). Using intracellular
protein from the filamentous fungus A. oryzae and the bacte-
rium E. coli, we show simple pretreatment of TCA precipitate
with mild alkali is rapid, requires addition of no potentially
interfering components, leads to significant solubilization of
* To whom correspondence should be addressed. Phone: (410) 455-3400.
Fax: (410) 455-1049. E-mail: marten@umbc.edu.
Table 1. Time Required for the Steps Involved in Various 2-DE
Protein Preparation Protocols
a Standard protocol4 involves addition of ªstandard sample solubilization
bufferº (SSSB), followed by vortexing for 2-4 h. b Sequential solubilization8
requires addition of a number of solubilizing agents, some of which have
been reported to interfere with 2-DE.1 c As described in Materials and
Methods, pretreatment of precipitate with approximately 20 íL 0.2 M NaOH
for 2 min. Volume then brought to 500 íL with SSSB. d Additional protein
may be solubilized if precipitate is frozen overnight, thawed, and vortexed
again requiring a total of approximately 24 h.
10.1021/pr025541x CCC: $25.00 xxxx American Chemical Society Journal of Proteome Research XXXX, X, XXXX-XXXX A
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protein precipitate, and does not reduce the number of visible
proteins or the quality of 2-D gels.
Materials and Methods
A. oryzae Growth and Cell Lysis Procedure. Wild type
Aspergillus oryzae (A1560; derived from strain IFO 4177,
institute for fermentation, Osaka, Japan) was obtained from
Novozymes North America, Inc. (Franklinton, NC). Storage,
media, and growth conditions for this strain have been
described previously.14 To lyse fungal cells, a mechanical cell
disruption method was employed.15 Briefly, the harvested
mycelial biomass (500 mg wet weight) and an equal amount
of acid washed glass beads (0.5 mm) were added to a 2 mL
Eppendorf tube containing 1 mL lysis buffer (20 mM Tris -
HCl pH 7.6, 10 mM NaCl, 0.5 mM deoxycholate, and 40 íL/ml
of protease inhibitor cocktail (Sigma Chemical Co, St. Louis,
MO). The mixture was agitated in a Mini-BeadBeater (BioSpec,
USA) at top speed, for 8 min (repeated cycles of 30 s on
followed by 30 s cooling on ice). The supernatant obtained after
centrifugation was then treated with DNase/RNase (Sigma
Chemical Co, St. Louis, MO). Finally, the supernatant divided
into two fractions (proteins from each fraction later used for
different types of resolubilization procedure) was further
treated with 20% v/v TCA (trichloroacetic acid, Sigma Chemical
Co, St. Louis, MO) in ice for 30 min to precipitate protein. The
precipitate was collected by centrifugation at 6000 g for 10 min
at 4 °C. The precipitated protein was washed with acetone to
remove traces of TCA and finally acetone was removed by
speed vacuum treatment.
E. coli Growth and Cell Lysis Procedure. E. coli wild type
strain W3110 was obtained from the Coli Genetic Stock Center
(CGSC; New Haven, CT). Storage, media, and growth conditions
for this strain have been described previously.16,17 Intracellular
protein sample for electrophoresis was prepared according to
the protocol from http://us.expasy.org/ch2d/protocols for E.
coli cells with the following variations in the lysis buffer (0.5
mM DTT instead of 0.5 mM DTE and 1.5 mM PMSF instead of
0.5 mM Pefabloc SC). After suspension in the lysis buffer, the
cells were lysed by sonication (550 Sonic Dismembrator, Fisher
Scientific, USA) at 40% power in ice. Cell debris was removed
by centrifugation, DNAse/RNAse were added to the super-
natant, and incubated for 30 min in ice. This protein sample
was divided into two fractions (proteins from each fractions
later used for different types of resolubilization procedure) and
subjected to 20% v/v TCA precipitation followed by acetone
washing and drying in speed vacuum similar to the A. oryzae
protein precipitation.
Protein Resolubilization. TCA precipitated protein pellets
of microbial intracellular proteins were solubilized by two
methods. In the first, TCA precipitate was resolubilized in 500
íl of standard sample solubilization buffer (SSSB; 8M Urea, 4%
w/v CHAPS, 1% w/v DTT; Sigma Chemical Co, St. Louis, MO)
and Ampholytes 2% v/v 3-10 nonlinear (NL; Amersham
Biosciences, San Francisco, CA, USA) alone. After 2-3 h of
intermittent vortexing, the sample was frozen at -80 °C for
approximately 15 h (overnight). The sample was then thawed
and subjected to an additional 2-3 h of vortexing (total
resolubilization time approximately 24 h). The insoluble mate-
rial was removed by centrifugation at 6000 g for 10 min. The
supernatant was collected and total soluble protein concentra-
tion measured (Plus one 2-D quantitation kit, Amersham
Biosciences, San Francisco, CA, USA, Cat. No. 80-6483-56). In
the second method, either 20 or 30 íl (for A. oryzae and E. coli
respectively) of 0.2 M NaOH was added to the TCA precipitated
pellet for 2 min., after which SSSB was added to make the final
volume 500 íl. The mixture was vortexed occasionally for 1-2
min, until the precipitate was completely solubilized. The total
soluble protein concentration was measured as above, and the
resulting solution was stored at -80 °C for later 2-DE.
2-D Electrophoresis. In preparation for 2-DE, 5 to 20 íL
(depending on protein concentration) of frozen protein sample
was diluted to 125 íL with rehydration solution.15 Rehydration
of Immobiline Dry Strips (IPG strip; Amersham Biosciences,
San Francisco, CA, USA) with sample was carried with an
Immobiline dry strip re-swelling tray (Amersham Biosciences,
San Francisco, CA, USA) according to manufactures instruc-
tions. IPG strips (pH 3-10 NL), 7 cm long were used for the
present study. The rehydrated strips were then subjected to
IEF. Isoelectric focusing was performed using Multiphor II
electrophoresis unit at 18 °C in gradient mode. Briefly, 7-cm
strips were focused at 0-200 V for 1 min, 200-3500 V for 1.30
h and 3500 V for 1.15 h, with a total of 8 kVh accumulated.
After focusing, the strips were stored at -80 °C for later use.
Prior to the second dimension SDS-PAGE, IPG strips were
equilibrated for 15 min in equilibration solution (5 mL)
containing 50 mM Tris-HCl pH 8.8, 6 M urea, 30% w/v glycerol,
2% w/v SDS, and traces of bromophenol blue with 100 mg/10
mL (w/v) of DTT. A second equilibration was carried out for
15 min by adding iodoacetamide (250 mg/10 mL) instead of
DTT in equilibration solution. A 5-mL portion of equilibration
solution was used for the 7-cm strip. Second dimension vertical
SDS-PAGE was performed using precast mini-gels (12% Tris-
HCl, 1 mm in thickness (Bio-Rad, CA). Mini-gel 2-DE was
Table 2. Quantitative Recovery of TCA Precipitated Proteins
(íg/íL) Solubilized by Different Methods
resolubilization method
organism SSSBa NaOH + SSSBb
increase in
soluble protein
A. oryzae 0.25 1.25 5.0�
E. coli 1.22 4.80 4.0�
a TCA protein precipitate solubilized with standard sample solubilization
buffer (SSSB) alone. After SSSB addition, precipitate vortexed approximately
3 h, frozen at -80 °C for 15 h and vortexed again approximately 3 h. b TCA
protein precipitate solubilized by pretreated with 0.2 M NaOH for 2 min
followed by addition of SSSB and vortexing for approximately 3 min.
Figure 1. Fraction of protein precipitate solubilized as a function
of 0.2 M NaOH pretreatment duration before addition of SSSB.
The total protein solubilized was 1.26 and 4.77 íg/íL respectively,
for A. oryzae and E. coli.
TCA Resolubilization technical notes
B Journal of Proteome Research
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carried employing Mini-vertical electrophoresis system (Mini
protean-II) according to the manufactures instructions. Briefly,
electrophoresis was performed at a constant current of 5 mA/
gel for 20 min, followed by 12 mA/gel for 1.5 hours until the
bromophenol band had exited the gel. Gels were stained with
neutral silver stain as described previously.18
Electropherogram images were obtained with an imaging
densitometer (GS-800; Bio-Rad) in gray scale mode. The image
analysis of gels was preformed using Z3 software (Compugen,
Israel; http://www.2dgels.com) as described previously.19 Briefly,
images of multiple gels, prepared with protein from each of
the two preparation protocols, were used to construct a
composite or ªraw master gelº (RMG). This serves to eliminate
noise and minor discrepancies between gels which should
theoretically be identical. The spots on the gels were then
identified, and each was assigned a spot quantity (q). The spot
Figure 2. 2-D electrophoresis of intracellular proteins from A. oryzae (A and B, 23 íg total protein loaded in each gel) and from E. coli
(C and D, 20 íg total protein loaded in each gel) resolubilized after TCA precipitation either with standard sample solubilization buffer
(SSSB) alone (A and C) or with NaOH pretreatment before addition of SSSB (B and D). Z3 software analysis on three-gel composites
shows 312, 328, 293, 309 total spots on gels A, B, C and D, respectively. Highlighted spots or regions call attention to significant
expression differences.
technical notes Nandakumar et al.
Journal of Proteome Research C
quantity characterizes spot size and intensity, is defined as the
sum of the gray-level values of all of the pixels in a spot, and
is expressed in terms of PPM (parts per million) of the total
spot quantity on the gel.20 Thus, q is an approximate fractional
representation of the amount of protein in a particular spot,
and the ratio of q values, for the same spot on two different
gels, gives an approximation of differential expression. All
molecular weight and pI values on gels were estimated using
protein markers obtained from Sigma Chemical Co (St. Louis,
USA; Cat. No. 6539) and Bio-Rad (CA, USA; Cat. No. 161-0310),
respectively.
Results and Discussion
Resolubilization of TCA Precipitated Proteins via Mild
Alkali Treatment. Intracellular proteins from both E. coli and
A. oryzae were extracted from whole cells and precipitated with
TCA as described in the Materials and Methods section. The
resulting protein precipitate was then resolubilized in one of
two ways: either (i) a standard sample solubilization buffer
(SSSB) was added directly to the protein precipitate or (ii) a
small amount of 0.2 M NaOH was added to the precipitate first,
followed by addition of SSSB. Results are shown in Table 2. In
initial tests we pretreated TCA precipitate with a number of
NaOH concentrations (0.2, 0.3, 0.5, 1.0 M; data not shown),
and found all worked relatively well for resolubilization. For
further testing, we choose the lowest concentration to avoid
potential problems with 2-DE. Figure 1 shows pretreatment
with 0.2 M NaOH for as little as 2 min was adequate to
significantly increase solubilization of protein precipitate from
both fungi and bacteria. In contrast, precipitate treated with
SSSB alone was not completely solubilized after incubation at
room temperature for 24 h, after freezing and thawing (i.e.,
overnight), or after these treatments combined with prolonged
vortexing. However, when this recalcitrant precipitate was
recovered via centrifugation and treated with 0.2 M NaOH, the
result was a significant increase in solubilization (data not
shown). Table 2 shows that compared to SSSB alone, pretreat-
ment with NaOH resulted in approximately 4 to 5 times more
soluble protein.
Comparison of Resolubilized Proteins via 2-D Electro-
phoresis. Efficiency of NaOH pretreatment for resolubilization
of TCA precipitate was evaluated using 2-DE (Figure 2). In
Figure 2, parts A and B, the same total amount of intracellular,
A. oryzae protein was loaded on each gel, whereas in Figure 2,
parts C and D, the same amount of intracellular, E. coli protein
was loaded. For both organisms, gels obtained with the two
different resolubilization protocols were generally comparable
with respect to the number and distribution of protein spots.
To determine whether resolubilization with NaOH pretreat-
ment affected the presence or intensity of individual spots, we
used Z3 image-analysis software. To remove noise and minor
discrepancies between nominally identical gels, three gels from
each pretreatment protocol were used to construct a composite
image (i.e., raw master gel). These composites were then
compared. For both organisms, we found a slightly higher
number of spots present on gels prepared with NaOH pre-
treated precipitate, than on those prepared with precipitate
resolubilized with SSSB alone (for A. oryzae 328 versus 312, for
E. coli 309 verses 293). To determine if individual proteins were
resolubilized differently by the two protocols, we used ªspot
quantityº (see Materials and Methods) to calculate protein
expression ratios (spot quantity of protein resolubilized with
NaOH pretreatment to spot quantity of protein resolubilized
with SSSB alone). Previous studies in our laboratory (on both
E. coli and A. oryzae) have shown protein expression ratios, on
gels from the same sample, can exhibit up to a 3-fold difference
(unpublished). Taking a conservative approach here, we note
only spots showing greater than 10-fold expression level
difference. Even with this constraint, Figure 3 shows for both
organisms twice as many spots showed increased expression
when precipitate was pretreated with NaOH than when pre-
cipitate was resolubilized with SSSB alone. It is interesting to
note that for fungal proteins, many of these proteins were
present in the acidic region of the gel. This was not the case
for the bacterial protein, were increased expression was most
strongly present in the center portion of the gel (Figure 2, parts
C and D, boxed area). We conclude that pretreatment with
NaOH for resolubilization of TCA precipitate yields comparable
2-D gels when compared to proteins resolubilized with SSSB
alone. In addition, NaOH pretreatment may slightly increase
the number of proteins resolubilized.
Others have reported that harsh treatments may lead to
interference during 1-D and 2-DE,1 and thus, treating proteins
with strong alkali or incubating for extended periods of time
at elevated pH, may lead to protein alterations, (e.g., modified
pI) or result in problems with electrophoresis. For this reason,
the protein precipitate in this study was treated with relatively
weak NaOH for a short period (2 min) and was then im-
mediately diluted approximately 25� with SSSB. Before IEF,
this mixture was further diluted with rehydration buffer. As a
result, the added NaOH was diluted approximately 250-fold
Figure 3. Logarithmic plot of protein expression ratios (spot
quantity for protein resolubilized with NaOH pretreatment, over
spot quantity for protein resolubilized with SSSB alone). Lines
drawn to illustrate 3� and 10� expression level differences. (A)
A. oryzae, 13 spots > 10� protein expression ratio, 5 spots <
0.1� protein expression ratio (B) E. coli, 27 spots > 10� protein
expression ratio, 11 spots < 0.1 � protein expression ratio.
TCA Resolubilization technical notes
D Journal of Proteome Research
before IEF, and we observed no problems typically associated
with salt accumulation (e.g., swelling or burning of IPG strips,
aggregation or precipitation of protein, etc.) in any of the runs.
In addition, serious smearing or horizontal streaking were not
evident in gels run on samples prepared with or without NaOH
pretreatment. We also note that 2-DE was performed on
samples from a number of different microbial growth stages,
and the results were highly reproducible (data not shown).
Quantitative loss of protein during solubilization of TCA
precipitate can be a problem in preparation of samples for
2-DE.15,21,22 Although an earlier report8 shows the resolubiliza-
tion of TCA precipitate is possible using a ªsequential solubi-
lizationº procedure, this method requires addition of a number
of components, in a multistep process, and requires ap-
proximately 2 to 3 h for completion.5,8 In contrast, the NaOH
pretreatment method presented here leads to a significant
increase in precipitate solubilization, requires addition of only
one component, and is complete in approximately 5 min, after
obtaining TCA precipitated proteins from lysed cell sample. Our
results with two different microbial systems imply that this
method is effective for prepar