ANALYTICAL BIOCHEMISTRY 170,491-494 (1988)

Enhanced Detection of Glycoproteins in Polyacrylamide Gels

G.M&OZ,S.MARSHALL, M.CABRERA,ANDA.HORVAT
Laboratorio de Genhica Molecular, Instituto de Biologia, Facultad de Ciencias B&cas y Matemriticas,
Universidad Cat6lica de Valparaiso. Valparaiso, Chile
Received September 14, 1987

A highly sensitive and simple method to enhance detection of glycoproteins resolved by either
one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a
modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F.
Kato, and G. Chappuis ( 1984) Arch. Viral. 80,69-82) that uses concanavalin A conjugated with
fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the
electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially
transferred to 50% methanol in deionized water and to absolute methanol. The result is an
abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement
of fluorescence is obtained as detected by exposing the treated gel to an appropiate uv source.
The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their
carbohydrate content in the range of 0.2 r.rg of total protein. The specificity of the detection is
demonstrated by a comparison with the corresponding polypeptide profile obtained by silver
nitrate staining of the gel. 0 1988 Academic F%s, Inc.

Several methods have been described to
detect glycoproteins in polyacrylamide gels
and most of them are either expensive or
time consuming (l-4). Notwithstanding
most of them employ a reliable and widely
accepted experimental procedure: the use of
different lectin molecules as tools for detec-
tion (5). In this report we describe a fast, in-
expensive, and highly sensitive method to
detect glycoproteins resolved in polyacryl-
amide gels, either in purified form or in bulk
cellular extracts, by enhancing fluorescence
of a conjugated-lectin probe after treatment
of the gel with absolute methanol.
MATERIALS AND METHODS
Fluorescent lectin probe. FITC-Con A’
was purchased from Sigma Chemical Co. (St.
Louis, MO).
Chemicals. Absolute methanol (analytical
grade) was purchased from Merck Laborato-
’ Abbreviations used IgG, human polyclonal immu-
noglobulin G; IgG, , mouse monoclonal immunoglobu-
lin G, ; Con A-FITC, lectin concanavalin A conjugated
with fluorescein isothyocyanate; SDS-PAGE, sodium
dodecyl sulfate-polyacrylamide gel electrophoresis.
ries (Germany). Acrylamide was from J. T.
Baker Chemical Co. (Phillipsburg, NJ), am-
pholites from Pharmacia (Uppsala, Sweden),
and SDS from Sigma Chemical Co. All other
reagents used were analytical grade. Solu-
tions were prepared in deionized water and
the acrylamide solution was further purified
using activated charcoal, deionizing resins,
and filtration.
Glycoproteins.Human polyclonal IgG was
purified in our laboratory by ammonium
sulfate precipitation followed by DEAE-col-
umn chromatography (6). Mouse BALB/c
monoclonal IgG, was obtained by the hy-
bridoma technique (7) at Universidad
Catoiica de Chile and was a kind gift from
Dr. A. de Ioannes.
Bulk cellular extracts. Bulk cellular ex-
tracts were obtained from surgical specimens
of human gastric cancer tissues directly ho-
mogenized in gel loading buffer and sub-
jected to electrophoresis.
Protein quantitation. Protein concentra-
tion was determined by the micro-Lowry
method (8).
Sample preparation for gel electrophoresis.
Proteins already in solution were mixed with

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Copyright Q 1988 by Academic Press, Inc.
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492 Mur;rOZ ET AL.

I 2 3 4 5 6 123456 I234 5 6

FIG. 1. Qualitative and quantitative analysis of IgG after SDS-PAGE. Lanes 1 through 5 contain 0.2,
0.4,0.6,0.8, and 1.O ~g of human polyclonal IgG. Lane 6 contains 1.O pg of mouse monoclonal IgG, . (A)
Photograph of the fluorescent gel after incubation with Con A-FITC. (B) Photograph of the fluorescent gel
after treatment with absolute methanol. (C) Photograph of the polypeptide profile on the gel after staining
with silver nitrate.

appropriate volumes of concentrated loading was transferred to a 50% methanol solution,

buffer as described by Laemmli (9) boiled at
100°C for 3-5 min, and immediately loaded
onto the gel. Volume sample ranged between
10 and 30 ~1.
For 2-D gels, proteins in solution were pre-
cipitated with 4-9 vol of cold absolute ace-
tone, incubated at -30°C for at least 30 min,
and centrifuged at 12,000g for 15 min, and
the pellet was resuspended in O’Farrell load-
ing solution (10).
Polyacrylamide gel electrophoresis. One-
dimensional gel electrophoresis was essen-
tially as described by Laemmli (9). Slabs of
10% polyacrylamide ( 14 X 11 cm and 0.5
mm thick) with a 3.5% stacking gel were elec-
trophoresed until the bromphenol dye
reached the bottom of the gel. For the 2-D
gel, the procedure was as described by
O’Farrell et al. (10). The first-dimension
electrofocusing was performed with ampho-
lites in the pH range 3.5-10. The second-di-
mension gel was as described above using the
same relation of stacking-to-resolving gel
concentration. In either case, after electro-
phoresis was completed, the gel was removed
from the glass plates, fixed and stained with
the fluorochrome-conjugated lectin as de-
scribed (1 l), and extensively washed to re-
move excess of free reagent. Then, the gel
and after 30 min of incubation at room tem-
perature it was immediately soaked in abso-
lute methanol until it turned completely
white and stiff. The fluorescence associated
with glycoproteins resolved in the gel was
detected before and after the methanol treat-
ment by means of an horizontal transillu-
minator (Chromato-Vue transilluminator,
Ultra-violet Products, Inc.) and photo-
graphed using Kodak plus-X film under a
yellow-orange filter. Finally, the gel was
transferred back to a fresh 50% methanol so-
lution and the polypeptide content was visu-
alized by the silver nitrate staining tech-
nique ( 12).
RESULTS AND DISCUSSION
In order to assay the enhancement of fluo-
rescence after methanol treatment of poly-
acrylamide gels in which variable amounts of
target proteins had been resolved, we selected
the monoclonal and polyclonal immuno-
globulin molecules as modified polypeptides
and Con A-FITC as the probe to detect their
carbohydrate moieties.
Figure 1 shows that for increasing concen-
tration of proteins fluorescence is specifically
detected associated with the heavy chain of
 ENHANCED DETECTION OF GLYCOPROTEINS IN ELECTROPHORESED GELS 493

JIH 3.5-10.0 pH 3.5-10.0

.

FIG. 2. Qualitative and quantitative analysis of IgG after 2-D electrophoresis. Thirty micrograms of
protein was applied on each of the first-dimension gels. Human polyclonal IgG is shown in (A-C). Mouse
monoclonal IgG, is shown in (D-F). (A) Photograph of the 2-D gel after exposure to Con A-FITC. (B)
Photograph of the fluorescent gel after treatment with absolute methanol. (C) Photograph of the gel after
staining with silver nitrate. (D) Photograph of the fluorescent gel at’ter staining with Con A-FITC. (E)
Photograph of the fluorescent gel after treatment with absolute methanol. (F) Photograph of the gel after
staining with silver nitrate.

either IgG (A). Nevertheless, fluorescence is
faint and not related to the amount of pro-
tein applied on the corresponding slot of the
gel. When the gel is treated with absolute
FIG. 3. Analysis of proteins and glycoproteins in bulk
cellular extracts after 10% SDS-PAGE. (A) Photograph
of the fluorescent gel after incubation with Con A-FITC
followed by incubation in absolute methanol. (B) Photo-
graph of the same gel stained with silver nitrate.
methanol, the intensity of fluorescence is in-
creased at least by a factor of 2-4 (Fig. 1B).
Therefore our method clearly enhances the
detection of the carbohydrate moiety of IgG,
and the specificity of recognition is verified
when both the heavy and light chains of the
IgGs are detected after silver staining the
same gel (Fig. 1C).
Since we worked with small concentra-
tions of single species of purified proteins, we
decided to try our method using 2-D gels
with higher amounts of the same proteins.
Figure 2 shows that for a 30-fold increase in
protein concentration the specificity of the
reaction is conserved, since only the heavy
chain of IgG fluoresces. Moreover, the 2-D
gel allows the resolution of a minor immuno-
globulin class contaminant in the polyclonal
human IgG of which as expected only the
heavy chain reacts with the fluorescent probe
(small dot in the upper right corner). Figure
494 MUIiIOZ ET AL.

2C shows the silver nitrate staining profile of species if the appropriate lectin probe is used
the same gel, where the light chain of the and/or for other protein-protein interac-
immunoglobulins lacking a carbohydrate tions, such as the recognition of antigen-an-
moiety appears as a diffuse band halfway tibody complexes. Also, since in our hands
down the gel, with no associated fluores- methanol treatment of silver stained gels en-
cence. As a control, a parallel 2-D gel was hances the resolution of protein bands, the
electrophoresed using the same concentra- method could offer and alternative approach
tion of monoclonal IgG (Figs. 2D, 2E, and to enrich detection of polypeptides already
2F). Results in Figs. 2A, 2B, 2D, and 2E resolved through polyacrylamide gel electro-
show the dramatic enhancement effect of phoresis. In conclusion, absolute methanol
absolute methanol when compared to the treatment of gels previously stained with
corresponding staining profiles in Figs. 2C fluorochrome-conjugated lectins offers an
and 2F. easy and fast way to characterize both quali-
Finally, we wanted to determine the speci- tatively and quantitatively the carbohydrate
ficity of the enhancement effect by analyzing content of a given protein sample using poly-
bulk cellular extracts. If any nonspecific in- acrylamide gel electrophoresis.
teraction would occur after exposure of the
gel to the lectin probe, the fluorescent profile ACKNOWLEDGMENTS
should be similar to that obtained after silver We thank Dr. James P. Robeson for critical analysis of
nitrate staining of the same gel. Figure 3 the manuscript and Mr. Carlos Gondez for preparing
shows that this is not the case. In Fig. 3A a the photographs. This research was supported by Dir-
heterogeneous glycoprotein profile is seen e&on General de Investigation, Universidad Cat&a
de Valparaiso, and by Fondo de Investigaci6n Cientifica
after methanol treatment of a gel enriched y Tecnol6gica de Chile, Fondecyt, Proyecto 35 l/87.
for a high concentration of polypeptides in
the range 40-60 kDa. In spite of this, the
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