Download Detection and characterization of gamete‐specific molecules in

RESEARCH ARTICLE
Molecular Reproduction & Development 76:4–10 (2009)
Detection and Characterization of Gamete-Specific Molecules
in Mytilus edulis Using Selective Antibody Production
HEIKO STUCKAS,1* KATRIN MESSERSCHMIDT,2 SASCHA PUTZLER,2 OTTO BAUMANN,3 JÖRG A. SCHENK,4
RALPH TIEDEMANN,1 AND BURKHARD MICHEEL2
1
Unit of Evolutionary Biology/Systematic Zoology, Institute of Biochemistry and Biology, University of Potsdam, Potsdam,
Germany
2
Unit of Biotechnology, Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany
3
Unit of Animal Physiology, Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany
4
UP Transfer GmbH, Hybrotec, Potsdam, Germany
SUMMARY
The mussel Mytilus edulis can be used as model to study the molecular basis of
reproductive isolation because this species maintains its species integrity, despite of
hybridizing in zones of contact with the closely related species M. trossulus or
M. galloprovincialis. This study uses selective antibody production by means of
hybridoma technology to identify molecules which are involved in sperm function of
M. edulis. Fragmented sperm were injected into mice and 25 hybridoma cell clones
were established to obtain monoclonal antibodies (mAb). Five clones were identified
producing mAb targeting molecules putatively involved in sperm function based on
enzyme immunoassays, dot and Western blotting as well as immunostaining of tissue
sections. Specific localization of these mAb targets on sperm and partly also in
somatic tissue suggests that all five antibodies bind to different molecules. The targets
of the mAb obtained from clone G26-AG8 were identified using mass spectrometry
(nano-LC-ESI-MS/MS) as M6 and M7 lysin. These acrosomal proteins have egg
vitelline lyses function and are highly similar (76%) which explains the cross reactivity
of mAb G26-AG8. Furthermore, M7 lysin was recently shown to be under strong
positive selection suggesting a role in interspecific reproductive isolation. This study
shows that M6 and M7 lysin are not only found in the sperm acrosome but also in male
somatic tissue of the mantle and the posterior adductor muscle, while being
completely absent in females. The monoclonal antibody G26-AG8 described here will
allow elucidating M7/M6 lysin function in somatic and gonad tissue of adult and
developing animals.
Mol. Reprod. Dev. 76: 4–10, 2009. ß 2008 Wiley-Liss, Inc.
Received 25 January 2008; Accepted 26 February 2008
INTRODUCTION
Molecules involved in reproductive traits are of interest in
evolutionary research as they are important to understand
mechanisms of reproductive isolation and speciation (e.g.
Vacquier et al., 1995; Singh and Kulathinal, 2000). Proteins
of functional importance in gametes have been studied in
ß 2008 WILEY-LISS, INC.
* Corresponding author:
Unit of Evolutionary Biology/
Systematic Zoology
Institute of Biochemistry and Biology
University of Potsdam
Karl-Liebknecht Strasse 24-25
Haus 26
14476 Potsdam (Golm), Germany.
E-mail: [email protected]
Published online 2 April 2008 in Wiley InterScience
(www.interscience.wiley.com).
DOI 10.1002/mrd.20916
many species. They were often found to evolve rapidly due
to positive Darwinian selection driven by various factors, that
is sexual selection, sperm competition, sexual conflict or
reinforcement (Swanson and Vacquier, 2002). Reinforcement is a mechanism causing prezygotic isolation as a result
of selection against hybrids (Dobzhanski, 1940). Free
spawning sessile marine invertebrates are an excellent
GAMETE-SPECIFIC MOLECULES IN MYTILUS EDULIS
model system to study the evolution of gamete proteins
since species recognition and hence reproductive isolation
can be assumed to almost entirely rely on gamete interaction. Here, proteins involved in acrosomal reaction are well
studied and were shown to be under strong positive selection such as bindin in sea-urchin species (Metz and Palumbi,
1996; Biermann, 1998; Geyer and Palumbi, 2003; Zigler
et al., 2003; McCartney and Lessios, 2007) or lysin and the
so-called 18-kDa protein in abalone species (Vacquier et al.,
1997; Kresge et al., 2001; Clark et al., 2007).
Marine mussels of the Mytilus edulis species complex (M.
edulis, M. trossulus, and M. galloprovincialis) can be used as
model to investigate reproductive isolation. These free
spawning bivalves occur worldwide in allopatric populations
but hybridize in zones of contact establishing stable hybrid
zones (Koehn, 1991). Assortative gamete interaction has
been identified as one factor causing reproductive isolation
in hybrid zones (Rawson et al., 2003) but its molecular basis
is not fully understood. A well studied candidate protein
involved in Mytilus gamete interaction is M7 lysin
(Springer and Crespi, 2007). It was identified by Tagaki et
al. (1994) as an acrosomal protein which has egg vitelline
lysis function and which can induce first polar body formation. Recent studies demonstrated that positive selection
shapes M7 lysin evolution (Riginos and McDonald, 2003;
Riginos et al., 2006; Springer and Crespi, 2007). Although
reinforcement could not be identified as a main factor causing selection pressure (Riginos et al., 2006), hybridization
and secondary contact are considered as a trigger for M7
lysin divergence between Mytilus species (Springer and
Crespi, 2007). In the light of these studies, it remains to be
further elaborated which molecules are involved in
sperm–oocyte interaction. In addition, it is of interest whether Mytilus gamete proteins including those that are not
involved in the acrosomal reaction are generally subject to
positive selection. Such investigations are however often
precluded, as proteins with particular gamete function remain to be characterized.
Experimental approaches based on selective antibody
€hler and
production by means of hybridoma technology (Ko
Milstein, 1975) can contribute substantially to discover,
isolate and characterize such tissue-specific factors. A similar approach was previously applied in other studies to
detect neuropeptides in M. edulis (Kellner-Cousin et al.,
1994) or to identify sperm-specific polypeptides in spermatozoa of marsupials (Harris and Rodger, 1998). The basic
principle is based on the injection of fragmented tissues into
mice, followed by establishing antibody producing hybridoma cell clones. These monoclonal antibodies (mAb) can
then be used for the identification and purification of tissuespecific target molecules and, in case of proteins, to identify
the corresponding gene. Furthermore, they can be applied
as experimental tools for functional target characterization.
This study aims to use selective antibody production to
identify factors involved in sperm function of M. edulis using
homogenized sperm as immunogen. mAb are raised and
tested for their specificity by analysing gametes and somatic
tissue of male and female specimens in enzyme immunoassays, protein blotting, and immunohistology. Particular antibody targets are identified by mass spectrometry.
Mol Reprod Dev 76:4–10 (2009)
RESULTS
Generation of Hybridoma Cell Clones and
Selection of mAb Preferentially Targeting
Male-Specific Proteins
Serum of mice which were immunized with fragmented
sperm of M. edulis were positively tested for the presence of
antibodies that bind to sperm but not to oocytes in enzyme
immunoassay. These animals were used to establish 25
hybridoma cell clones that were successfully screened for
monoclonal antibody (mAb) production. To test binding
specificities of these antibodies, they were applied to native
protein extracts of sperm and oocytes in enzyme immunoassay and dot blot assays. Furthermore, they were tested for
binding to denatured protein extracts from gametes, mantle,
and foot tissue of males and females in Western blots (see
Fig. 1 for an example) and the antibody subclasses were
determined. Based on these initial assays, five antibodies
were found to bind targets preferentially occurring in males
(G26-DD5, G26-DA10, G26-AG8, G26-EB3 and G26-DH5;
Table I). The G26-DA10, G26-AG8, G26-DH5 preferentially
bound to native (enzyme immunoassays, dot blot) and
denatured (Western blot) protein extracts of sperm. However, antibody G26-DH5 showed a weak binding to native
oocyte extracts in enzyme immunoassays. All three mAb
partly bound to protein extracts of male somatic tissue in
Western blot (G26-DA10, G26-AG8, G26-DH5 to mantle;
G26-DH5 to foot; G26-AG8 to posterior adductor muscle).
Antibody G26-DD5 bound native sperm protein extract in
enzyme immunoassays and dot blot whereas antibody G26EB3 only bound to sperm extracts in enzyme immunoassays. The fact that both antibodies (G26-DD5, G26-EB3) did
not bind to a target detectable by Western blotting could
indicate that the detected epitope is specific for native
proteins (discontinuous epitope). All other 20 antibodies did
not show any sex and/or tissue-specific binding pattern in
Figure 1. Western Blot to demonstrate tissue and gender specificity of
the mAb G26-AG8 target M6/M7 lysin in somatic tissue and gametes.
A: Protein extracts obtained from foot, muscle (posterior adductor
muscle), and mantle somatic tissue of males ( ) and females ( ).
B: Protein extracts obtained from sperm and oocytes. Actin was
detected as loading control in all procedures. Note that similar
amounts of actin were detected in each tissue type in both sexes
indicating equal loading of tissue-specific protein extracts.
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STUCKAS
ET AL.
TABLE I. Description of Target Localization Derived From Histological Analysis
Target localization
Gonade
Antibody
G26-DD5
G26-DA10
G26-AG8
G26-EB3
G26-DH5
Mantle
Subclass
IgG2a
IgG2b
IgG2a
IgG2b
IgG1b
Whole sperm surface
Sperm tail
Acrosome
Gonad soma
Whole sperm surface
Epithelial cells
—
—
—
Epithelial cells
—
Muscle cells
—
—
—
—
Muscle cells
—
—
—
Note that no specific binding of any mAb to foot or posterior adductor muscle tissue was found in males or females. (—) Indicates nonbinding of antibodies.
these assays and were excluded from further investigation in
the study presented here.
Analysis of mAb Target Localization
To analyse further the specificity of the pre-selected
antibodies, sections of gonads and somatic tissue
(mantle, posterior adductor muscle, foot) of male and female
specimens were investigated using immunohistochemical
approaches (Fig. 2). These results are summarized in
Table I.
The target of antibody G26-AG8 was found only in sperm
(Fig. 2C). Based on the ultrastructural characterization of M.
edulis sperm by Nijima and Dan (1965) the target of this mAb
was found to be specific to the acrosome and appears to be
located in a region termed as ‘partition bounding basal ring’.
The target of antibodies G26-DD5, G26-DA10, G26-DH5
could also be detected on particular structures of spermatozoa (Fig. 2) but also in some somatic tissue (Table I) while
being absent in oocytes. Antibodies derived from the
hybridoma cell clone G26-DD5 bound to a molecule homogeneously distributed on the entire surface of spermatozoa
as well as in epithelial cells of female gonads. Similarly, the
target of mAb G26-DA10 has a scattered distribution on
the entire sperm and is also found in epithelial cells of the
ovary. The target of mAb G26-DA10 seems to be specific to
the sperm tail but is also present in somatic cells of the
mantle in males and females. Finally, the target of mAb G26EB3 was found in male gonads, but could not be found in a
particular structure of spermatozoa.
Analysis of the Target Protein of mAb G26-AG8
To demonstrate the applicability of our approach to identify the primary structure of gamete proteins, the target of
mAb G26-AG8 was analysed using mass spectrometry. This
choice was based on the fact that the acrosomal localization
of this target molecule suggests an involvement in the
fertilization process and hence in reproductive isolation of
M. edulis. To isolate the target protein, whole sperm protein
extracts were separated using two-dimensional gel electrophoresis. As shown in Figure 3, two target protein spots were
identified and analysed using mass spectrometry. The sequences of peptides were analysed and allocated to known
proteins using the MASCOT search engine and nr protein
databases (Table II). One protein spot was identified as
6
vitelline coat M6 lysin from M. edulis (probability score of
216). Furthermore, the second protein spot was identified as
vitelline coat M7 lysin matching protein database entries
originating from M. galloprivincialis (five entries; probability
scores from 135 through 175) and M. edulis (four entries;
probability scores from 165 through 167). Both proteins were
initially described after isolation from acrosomes of M. edulis
sperm (Tagaki et al., 1994).
DISCUSSION
This study used selective antibody production to establish five mAb binding targets localized in particular structures
of spermatozoa and male gonads of M. edulis. None of these
targets were identified to occur in oocytes of this species, but
some are present in other male and some female somatic
tissues. These five mAb were selected out of 25 antibodies.
The properties of the remaining 20 mAb which are not
specific to a particular tissue type will be reported elsewhere.
In order to demonstrate that our approach of selective
antibody production can identify factors directly involved in
reproductive traits, one selected target was analysed using
mass spectrometry. This procedure showed that the mAb
G26-AG8 binds the two proteins M6 and M7 lysin. These
proteins were originally identified by Tagaki et al. (1994) to
lyse egg vitelline and to release the first polar body. Both
proteins are located in the acrosome and have a molecular
weight of approx. 20 kDa. In fact, this study shows that the
target of mAb G26-AG8 is localized in the acrosome and
both one- and two-dimensional protein electrophoresis suggest a molecular weight of approx. 20 kDa. The cross
reactivity of mAb G26-AG8 with both M6 and M7 lysin can
be attributed to the high similarity of 76% of both proteins as
determined by Tagaki et al. (1994). Since no antibodies were
available so far for these Mytilus proteins, mAb G26-AG8
can be used to elucidate M6/M7 lysin function in adult and
developing specimens. Our investigation can, therefore,
extend the knowledge about both proteins in M. edulis. We
provide evidence that M6/M7 lysin has a particular localization in an acrosome region named as ‘partition bounding
basal ring’ by Nijima and Dan (1965) which contains the
‘basal ring material’. Furthermore, Western blot analysis
suggests the occurrence of one or both proteins in male
mantle and muscle tissue which raises the question whether
M6 and M7 lysin have functions in addition to egg vitelline
Mol Reprod Dev 76:4–10 (2009)
GAMETE-SPECIFIC MOLECULES IN MYTILUS EDULIS
Figure 2. Localization of antibody targets on sperm as analysed with
fluorescent microscopy (A1–E1), differential interference contrast
microscopy (A2–E2) and overlay pictures (A3–E3). The target of mAb
G26-DD5 (A1–A3) is homogenously distributed on the entire sperm
and the target of mAb G26-DA10 (B1–B3) was found exclusively on
the sperm tail. The mAb G26-AG8 (C1–C3) targets a protein localized
in the acrosome and arrows indicate the position of the acrosome (Ac)
and the putative position of the tail (T). No particular localization on
spermatozoa could be identified for the target of mAb G26-EB3. A
target showing a scattered distribution on the sperm surface is bound
by mAb G26-DH5. The bar represents a scale of 10 mm.
coat lysin and first polar body releasing activity. Since our
histological study could not identify any particular localization of M6/M7 lysin within the mantle or posterior adductor
muscle, this question has to be addressed in future studies.
However, our observation could partly be explained by the
fact that spermatogenic tissue ramifies throughout the mantle (personal communication; T. Bartolomaeus, Free University Berlin, Germany).
Mol Reprod Dev 76:4–10 (2009)
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STUCKAS
ET AL.
also been detected in various types of somatic tissue.
However, future experiments have to clarify the nature of
the target molecules of the other antibodies. Since our
approach of using selective antibody production led to the
identification of a known protein complex, it can also be
expected that completely unknown proteins will be identified
(i.e. as a result of de novo sequencing of peptides by mass
spectrometry).
Figure 3. Coomassie Brilliant Blue stained gel obtained by 2-DE of
sperm protein extracts based on isoelectric focussing (first dimension,
pH3-pH11) and SDS–PAGE (second dimension, molecular weight
10–150 kDa). Western Blotting of a replicate gel revealed protein
spots representing M6 lysin and M7 lysin as targets of mAb G26-AG8.
M7 lysin is one target of our newly described mAb AG8A8. This protein has been demonstrated to be under strong
positive selection (Riginos and McDonald, 2003) although
the underlying evolutionary mechanisms are still under
debate (reinforcement or selection pressure caused by
secondary contact; Riginos et al., 2006; Springer and Crespi, 2007). Here, our new mAb G26-AG8 can stimulate
further research on M7 lysin (including the analysis of its
functional importance in mantle and muscle tissue) and
might contribute to a better understanding of selective forces
driving the evolution of this protein.
Despite the remaining four target molecules were not fully
identified yet, the following characteristics have been
already revealed in this study: The different localization of
mAb targets on sperm count for the fact that all five antibodies bind to different target molecules (Fig. 2). Despite the
fact that the antibody targets are localized in spermatozoa,
their functional relevance might not necessarily be restricted
to sperm. This was concluded from the fact that they have
CONCLUSIONS
The study demonstrated the applicability of selective
antibody production to detect sperm-specific factors. During
the course of this study, five mAb were selected binding
particularly targets located on sperm. The targets of one
monoclonal antibody (G26-AG8) have been characterized
by mass spectrometry as M6 and M7 lysin. These proteins
were localized in an acrosomal region called ‘partition
bounding basal ring’ and found in somatic tissue (mantle,
posterior adductor muscle). This newly described antibody
(G26-AG8) is applicable in future investigations, that is to
analyse Mytilus development or to understand the molecular
basis and evolution of reproductive processes. We argue
that selective antibody production has some remarkable
characteristics compared with pure genetic approaches:
(1) The resulting antibodies can be used to perform additional functional studies (i.e. search for sperm–egg protein
interaction through co-immunoprecipitation). (2) Since protein targets of mAb’s can be directly sequenced (i.e. de novo
sequencing by mass spectrometry), the approach can principally be used without any prior knowledge of the genome.
This makes it particularly attractive for the application to
nonmodel organisms. (3) Antibodies have the ability to
detect functional important secondary modifications of proteins (e.g. glycosylation and/or phosphorylation patterns).
MATERIALS AND METHODS
Tissue Preparation
M. edulis specimens were collected from the Baltic Sea coast at
Kiel (Germany). Sex determination was performed by microscopic
TABLE II. Analysis of mAb G26-AG8 Target Proteins (Compare Fig. 3) Using Nano LC-ESI-MS/MS Followed by MASCOT
Database Search
Identified protein
NCBI acession
number Protein
Vitelline coat lysin M6
precursor of M. edulis
gi 28630406
Vitelline coat lysin M7
precursor of
M. galloprovincialis and
M. edulis
gi 28630368
Mr (exp)*
Mr (calc)†
Matched peptide sequence‡
2020.96
1894.23
2192.59
1987.25
1454.07
2031.91
1750.11
2004.13
1466.11
1599.11
2021.99
1893.89
2192.05
1986.98
1453.66
2032.94
1750.85
2004.00
1465.70
1597.74
20–37: KSNGNGYIYINHVTGETR
21–37: SNGNGYIYINHVTGETR
38–60: TSPPTHGSSGSAPAPAQISASER
98–113: GELFWPDLPYESFFLK
121–132: TSTHFFWTNGEK
133–150: HNGQWNWGTGHPAFTAPR
14–28: MNGFIYINHVTGETR
29–49: TSPPTHGSSGTGPAPVQISAR
112–123: ISTHFFWTNGEK
128–141: WNWGTGHPAFSNPK
*Experimental m/z transformed to a relative molecular mass.
†
Relative molecular mass calculated from matched peptide sequence.
‡
Position of the peptide within identified protein is indicated by the corresponding amino acid number.
8
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GAMETE-SPECIFIC MOLECULES IN MYTILUS EDULIS
examination of gonads for the presence of sperm or eggs. Gametes
were isolated by placing scored gonad tissue into filtered sea water.
After incubation (approx. 1 hr) the supernatant was centrifuged to
pellet the gametes which were subsequently frozen in liquid nitrogen and stored at 80 C. Tissue samples (gonad, mantle, posterior
adductor muscle, foot) were excised, rinsed in phosphate buffered
saline (PBS) and transferred to an appropriate fixation buffer for
histological analysis (see below) or frozen in liquid nitrogen and
stored at 80 C for protein extraction.
Protein Extraction
Frozen tissue was homogenized under liquid nitrogen. For
subsequent use in enzyme immunoassay, dot and Western blotting, mass-equivalent amounts of lysis buffer (0.5 M Tris–HCl pH
6.8 with 2.8% SDS, 10% glycerol and 0.5% b-mercaptoethanol)
were added and the samples were incubated for 10 min at 95 C
followed by centrifugation (16,000g, 5 min, room temperature).
Alternatively, for application in 2 D electrophoresis, tissue homogenates were incubated in a lysis buffer (7 M urea, 2 M thiourea, 2%
carrier ampholytes, 70 mM DTT) for 30 min at room temperature
followed by centrifugation (10,000g, 30 min, room temperature).
Supernatants obtained in both approaches were transferred to new
reaction tubes and stored at 80 C. Protein concentrations were
determined by Bradford assays.
Generation of Monoclonal Antibodies
Isolated M. edulis sperm were fragmented through repeated
freezing and thawing. Male C57/Bl6 mice were immunized intraperitoneally with 25 ml of these sperm preparations with 50 ml
complete Freunds adjuvant. Mice were boosted with 10 ml antigen
in 100 ml PBS after 10 weeks. Six days later, sera were tested for
sperm-specific antibody levels in enzyme immunoassay. Responding mice were boosted again with 10 ml antigen in 100 ml PBS. Four
days later spleen cells of the mice were fused with Sp2/0-Ag14
myeloma cells (ATCC: CRL-1581) using a modified electrofusion
technique (Schenk et al., 2004). Briefly, the spleen/myeloma cell
ratio was about 3:1 in 10% PEG 8000 and the voltage ranged from
3,000 to 3,500 V/cm. Following fusion, the cells were plated into 96well plates (Nunc, Wiesbaden, Germany) and cultured in RPMI
1640 medium containing 10% fetal calf serum and hypoxanthineazaserine-thymidine (HAT). Selected hybrids were cultivated,
cloned by limiting dilution, and stored in liquid nitrogen according
to standard methods.
Enzyme Immunoassays
Microtitration plates were coated either with fragmented sperm
for mouse serum analysis or with native protein extracts for analysis
of hybridoma cell culture supernatant. Hereby, 50 ml of sperm or
protein solution was incubated at 4 C overnight prior to washing
with tap water and blocking with 50 ml PBS/NCS (neonatal calf
serum) per well for 1 hr at room temperature. Mouse serum (50 ml/
well, pre-incubated with fragmented oocytes to select sperm-specific antibodies) or hybridoma cell culture supernatant (50 ml/well)
were incubated for 1 hr followed by washing with tap water. To
detect bound murine antibodies, a peroxidase labelled goat-antimouse IgG antibody (Dianova, Hamburg, Germany) was applied
and tetra-methyl-benzidine (TMB) solution (0.12 mg/ml TMB with
0.04% hydrogen peroxide in 25 mM NaH2PO4) was used
as substrate. The reaction was stopped with 1 M H2SO4 after
5–10 min and measured at 450 nm in a microtitration plate
reader. To determine antibody subclasses, the same procedure
was performed except that biotin labelled goat anti-mouse immunoglobulin isotype-specific antibodies (Serva, Heidelberg,
Germany) were used as secondary antibodies and streptavidinperoxidase conjugate (Roche, Mannheim, Germany) and TMB
solution as indicators.
Mol Reprod Dev 76:4–10 (2009)
Gel Electrophoresis and Immunoblotting
For one-dimensional electrophoresis (1-DE) of proteins, samples (10 mg protein/lane) were loaded onto sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS–PAGE) gradient gels
(7.5–15%) under reducing conditions. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Protran BA 83,
€ll, Dassel, Germany). For
pore size 0.2 mm, Schleicher and Schu
immunodetection, membranes were blocked in PBS containing
1% (w/v) BSA for 1 hr at room temperature and then incubated
with culture supernatants of the generated antibodies [diluted
1:2 in PBS containing 1% (w/v) BSA] for 2 hr at room temperature.
To demonstrate equal loading, the mouse anti-chicken actin
IgG [ICN Biomedicals, Costa Mesa, CA; diluted 1:400 in
PBS containing 1% (w/v) BSA] was used and membranes
were incubated for 2 hr at room temperature. The membrane was
then incubated with peroxidase-conjugated goat anti-mouse IgG for
1 hr at room temperature and developed with diaminobenzidine
(DAB) solution as substrate [100 mM Tris–HCl pH 7, 0.08% (w/v)
DAB, 0.04% (w/v) NiCl2, 0.01% H2O2]. Between all incubation steps
the membrane was washed with PBS supplemented with 0.1%
Tween-20.
For dot blotting native protein extracts were dropped onto a
nitrocellulose membrane (as used for 1-DE Western blotting).
Immunodetection was performed as described for Western
Blotting.
Two-dimensional electrophoresis (2-DE) was performed following the protocol by Klose and Kobalz (1995). Isoelectric focussing
(IEF; first dimension) was performed in vertical rod gels containing 9
M urea, 4% acrylamide, 0.3% piperazine diacrylamide, 5% glycerine, 2% carrier ampholyte (pH 2-11), 0.06% TEMED, 0.08% ammonium persulfate. The total amount of 30 mg of protein was
processed and focussed at 1,841 V h. Subsequently, IEF gels were
incubated in a buffer containing 125 mM Tris-phosphate (pH 6.5),
40% glycerol, 65 mM dithiothreitol (DTT), and 3% SDS for 10 min.
After this equilibration step, gels were frozen at
20 C.
SDS–PAGE (second dimension) was performed in gels [0.1 cm 20 cm 30 cm; 15% acrylamide, 0.2% bisacrylamide, 375 mM
Tris–HCl (pH 8.8), 0.1% SDS, 0.03% TEMED, 0.08% ammonium
persulfate]. Frozen IEF gels were thawed, applied to SDS gels, and
covered with 0.5% agarose. Electrophoresis was performed at
150 V and 30 mA for 75 min. Two replicate 2-DE separations were
performed. One gel was stained with Coomassie Brilliant Blue for
preparative applications; the other gel was used for Western
blotting to detect the target protein by immunostaining.
Blotting of 2-DE gels was performed using an Immobilon-P
membrane (PVDF, pore size 0.45 mm; Millipore, Bedford, MA) and
€nchen,
a Trans-Blot SD Semi-Dry Transfer Cell (Biorad, Mu
Germany) at a constant current of 1 mA/cm2 and 80 V for 2 hr at
4 C using a blotting buffer consisting of 25 mM Tris–HCl, 192 mM
glycine, 0.1% SDS (pH 8.3) and 20% methanol.
For immunodetection of proteins, membranes were washed in
TBST [20 mM Tris–HCl (pH 7.5); 154 mM NaCl, 0.1% Tween-20]
and blocked in TBST containing 2% (w/v) BSA for 2 hr. Membranes
were incubated in culture supernatant of mAb clone G26-AG8
[diluted 1:3 in TBST containing 1% (w/v) BSA] overnight and then
incubated with alkaline phosphatase conjugated goat anti-mouse
IgG (Sigma, Taufkirchen, Germany) [diluted 1:10,000 in TBST
containing 1% (w/v) BSA] for 1 hr at room temperature. Finally,
bound antibodies were detected by incubating with Fast Red/
Naphtol (Sigma) for 5 sec. Between all incubation steps the membrane was washed in TBST (5 times for 10 min).
Identification of 2-DE Separated mAb Target Proteins
Protein identification using nano LC-ESI-MS/MS was performed
by the Proteome Factory (Proteome Factory AG, Berlin, Germany;
http://www.proteomefactory. com). The MS system consists of an
Agilent 1100 NanoLC system (Agilent, Santa Clara, CA), PicoTip
9
Molecular Reproduction & Development
emitter (New Objective, Woburn, MA) and an Esquire 3000 plus ion
trap MS (Bruker, Bremen, Germany). Protein spots were in-gel
digested using trypsin (Promega, Madison, WI) and applied on a
column (Zorbax SB C18, 0.3 mm 5 mm, Agilent) using 1%
acetonitrile/0.1% acetic acid for 5 min. Desalted peptides were
applied on a column (Zorbax 300 SB C18 column, 75 mm 150 mm,
Agilent) and separated through a gradient reaching from 5%
acetonitrile/0.1% acetic acid to 40% acetonitrile/0.1% acetic acid
within 40 min. MS spectra were automatically taken by Esquire
3000 plus according to manufacturer’s instrument settings for nano
LS-ESI-MS/MS analyses. Proteins were identified using MS/MS
ion search of MASCOT search engine (Matrix Science, London,
UK) and nr protein database (National Center for Biotechnology
Information, Bethesda, MD). MASCOT expresses the probability
that peptides match at random to a given protein by a probability
score. A score larger than 57 indicates identity or extensive homology (P < 0.05).
Immunohistochemistry and Confocal
Laser Scanning Microscopy
Freshly excised tissue was fixed with 3% formaldehyde in PBS
for 1 hr, washed 3 times for 10 min in 0.1 M phosphate buffer pH 7,
incubated in 10% sucrose in PBS for 1 hr and finally infiltrated with
25% sucrose in PBS overnight at 4 C. The specimens were shockfrozen in isopentane, cooled to approx. 150 C, and stored at
80 C. Tissue slices of 5–20 mm thickness were generated with a
cryostat (Microm HM 500 OM, Microm International GmbH, Walldorf, Germany) at 30 C, dried on glass slides for 30 min at room
temperature and stored at 80 C. Before use, tissue slices were
treated with methanol/acetone (1:1) for 10 min at 20 C, dried at
room temperature for 10 min, and incubated in 50 mM ammonium
chloride in PBS. After washing in PBS for 5 min unspecific binding
was blocked with PBS containing 10% neonatal goat serum (NGS)
for 1 hr at room temperature. Undiluted culture supernatants containing the mAb were added and incubated overnight at 4 C. After
washing 3 times for 5 min with PBS, bound antibody was detected
with Cy3-labelled goat anti-mouse IgG antibody [Dianova; diluted
1:50 in PBS containg 10% (w/v) NGS] by incubation for 1 hr at room
temperature. Specimens were mounted in Mowiol 4.88 supplemented with 2% n-propyl-gallate as an anti-fading reagent. Omitting the
first antibodies served as control for specificity. Specimens were
examined with a confocal laser scanning microscope (LSM 510,
Carl Zeiss, Jena, Germany).
ACKNOWLEDGMENTS
€rbel May for technical support. The authors are
We thank Ba
grateful to Thomas Bartolomaeus for discussion of results obtained
from immunostaining of tissue sections. We are thankful to Carola
Lehmann and Christian Scheler from Proteome Factory AG, Berlin,
Germany for mass spectrometry analysis.
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