FR AB - Science Reference

ABRF 2001 ABSTRACTS
P129-T
Isolation and identification of rat liver proteins using ultracentrifugation
with Nycodenz and 1D/2D-SDS-PAGE.
K. Murayama1, T. Fujimura1, M. Morita2, N. Shindo1; 1Juntendo Univ. Sch.
of Med., 2-1-1, Hongo, Bunkyo-ku, Tokyo, Tokyo 113-8421, Japan, 2Hitachi
Koki Co., Ltd.
The use of 2D-SDS-PAGE as a clinical molecular scanner of various tissues
and physiological fluid samples has proved useful. However, each organ contains
more than 4,000 proteins and accordingly, it is almost impossible to
study the functional role of these proteins unless separated. Our ultimate goal
is to use 2D-SDS-PAGE as a clinical molecular scanner to define each
organelle in various organs.
In this study, we report the isolation of rat liver organelles by density gradient
centrifugation using Nycodenz. Nycodenz solution at 10, 20, or 30% concentration,
containing 0.25 M sucrose (as an osmotic balancer), was added
to each centrifuge tube and allowed to stand overnight at �20 to �80�. The
solution was thawed at room temperature (�2 h), and analyzed to construct
a density gradient curve. When used in a 5-ml tube, Nycodenz gradient densities
from the top to the bottom without any centrifugation were 1.0334 to
1.2188 at 10%, 1.0506 to 1.2878 at 20% and 1.0856 to 1.3199 at 30%. Liver
homogenate (0.4 ml, 4 mg) was loaded on the Nycodenz gradient solution
and centrifuged at 28,000 rpm for 20 min using a Hitachi ultracentrifuge
CP100�-RPS40T-2. The mixture was fractionated by a fractionator, DGF-U, its
absorbance measured at 360 nm with a spectrophotometer and density with
an Abbe refract meter. Next, 5 �l of each fraction was applied onto 10% gel
for 1D-SDS-PAGE, electrophoresis, and the gel was stained by silver nitrate.
Another 1D-SDS-PAGE was erector-blotted to a PVDF membrane and the
presence of organelles was confirmed using antibody of the marker protein
for each organelle.
P131-M
Selective depletion of major serum proteins and fractionation prior
to 2-dimensional differential gel electrophoresis.
J.J. Cummings, E. Rohde, P.R. Griffin; Merck Res. Labs., RY800-B210, Rahway,
NJ 07065
Two dimensional differential gel electrophoresis (2DIGE) is a powerful technique
for the study of protein expression in physiological fluids such as
serum. However, the presence of a few major proteins interferes with the
separation and detection of many low abundant, yet physiologically important
proteins. Albumin (52%), IgG (20%), IgA (2.5%), IgM (1.6%), transferrin
(3.6%) and �1-antitrypsin (1.6%) are the major constituents of serum in many
species.
Our objective was to selectively remove abundant proteins in a few sequential
steps followed by the division of the depleted sera into multiple fraction
based on their hydrophobicity (RP-HPLC) and/or charge (IEX-HPLC) prior to
pre-electrophoresis fluorescent labeling.
The effective removal of albumin was accomplished using Cibachron blue
dye spin columns. Immunoglobulins (IgG, IgM) were removed by gel filtration
over protein A and G columns. Affinity resins specific to transferrin and
�1 antitrypsin were prepared in house and used for the depletion of the
respective proteins. Chromatographic fractionation was carried out on largebore
columns (4.6 � 100 mm). During all depletion and fractionation steps
emphasis was placed on maximizing protein recovery. The protein concentration
was monitored spectrophotometrically and effectiveness of depletion
was assessed by SDS-PAGE.
The separation of the depleted and fractionated sera by 2DIGE resulted in a
significant increase in the dynamic range of the separation. Utilizing this
approach proteins were detected in areas previously obscured by major
serum constituents. Furthermore an increased number of proteins was
observed. Combined with the power of differential protein mapping using
fluorescent dyes the procedure has shown great utility in the search for differentially
expressed proteins. We present examples of coupling 2DIGE with
�LC-MS/MS and database searching for the identification of surrogate markers
in sera.
POSTER ABSTRACTS
220 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P130-S
Rapid and simple single nanogram detection of glycoproteins
in polyacrylamide gels and on electroblots.
W.F. Patton, T.H. Steinberg, K.N. Berggren, K. Pretty On Top, C. Kemper,
Z. Diwu, R.P. Haugland; Molecular Probes Inc., 4849 Pitchford Avenue,
Eugene, OR 97402
The fluorescent hydrazide, Pro-Q Emerald 300 dye, may be conjugated to
glycoproteins by a periodic acid Schiff’s (PAS) mechanism. The glycols present
in glycoproteins are initially oxidized to aldehydes using periodic acid.
The dye then reacts with the aldehydes to generate a highly fluorescent conjugate.
Reduction with sodium metabisulfite or sodium borohydride is not
required to stabilize the conjugate. Though glycoprotein detection may be
performed on transfer membranes, direct detection in gels avoids electroblotting
and glycoproteins may be visualized 2–3 hours after electrophoresis.
This is substantially more rapid than PAS labeling with digoxigenin
hydrazide followed by detection with an anti-digoxigenin antibody conjugate
of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by
detection with horseradish peroxidase or alkaline phosphatase conjugates of
streptavidin, which require more than eight hours to complete. Pro-Q Emerald
300 dye is spectrally compatible with SYPRO Ruby protein gel stain,
allowing two-color detection of glycosylated and nonglycosylated proteins on
the same gel or blot. Both fluorophores are excited with mid-range UV illumination.
Pro-Q Emerald 300 dye maximally emits at 530 nm (green) while
SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of a1acid
glycoprotein (40% carbohydrate) and 1 ng of avidin (10% carbohydrate)
or glucose oxidase (12% carbohydrate) are detectable in gels after staining
with Pro-Q Emerald 300 dye. Besides detecting glycoproteins, as little as 2–8
ng of lipopolysaccharide is detectable in gels using Pro-Q Emerald 300 dye
while 250–1000 ng is required for silver staining. Detection of glycoproteins
may be achieved in 1-D or 2-D gels and on PVDF or nitrocellulose membranes.
P132-T
Rice tissue proteomics: towards a functional analysis of the
rice genome.
S. Komatsu, S. Shen, Z. Li, G. Yang, H. Konishi, M. Yoshikawa, R. Rakwal;
Natl. Inst. of Agrobiol. Resources, 2-1-2 Kannondai, Tsukuba, Ibaraki
305-8602, Japan
The technique of proteome analysis with two-dimensional polyacrylamide
gel electrophoresis (2D-PAGE) has the power to monitor global changes that
occur in the protein expression of a tissue, an organism, and/or under
stresses. In this study, proteins extracted from endosperm, embryo, root, callus,
green shoot, etiolated shoot, leaf sheath and panicle of rice were separated
by 2D-PAGE. The separated proteins were electroblotted onto a
polyvinylidene difluoride membrane. The N-terminal amino-acid sequences
of 117 out of 377 proteins were determined in this manner. N-terminal
regions of the remaining proteins could not be sequenced and they were
inferred to have a blocking group at N-terminus. Internal amino-acid
sequences of 260 proteins were determined using the protein sequencer or
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
after enzyme digestion of proteins. Finally, a data-file of rice proteins was
constructed, which included information on amino-acid sequence and
sequence homology. Using this experimental approach, we could identify the
major proteins involved in growth and development of rice. Some of these
proteins, including a calcium-binding protein, which turned out to be carleticulin
in rice, have functions in signal transduction pathway. The information
thus obtained from amino-acid sequence of these proteins will be
helpful in predicting the function of the proteins and for their molecular
cloning in future experiments.
This work was supported in part by a grant of Rice Genome Project PR-1201,
MAFF, Japan.