Purification and identification of zebrafish Vtg
The elution profile of WBH from E2-treated males on Sephacryl S-300 column
showed a UV-absorbing peak at the elution volume between 70 and 85 mL (Figure
S1A), whereas no peak was observed at the corresponding position for the control
WBH. Fractions of this peak were further subjected to anion exchange
chromatography, and a main peak was detected at 0.2 M NaCl (Figure S1B).
Figure S1. (A) Elution profiles of whole body homogenate from control and
E2-treated
males
on
a
Sephacryl
S-300
column.
(B)
DEAE-Sepharose
chromatography of Vtg fractions from gel filtration (the second dashed-line peak in
Fig 1A).
In native-PAGE, two major protein bands with low electrophoretic mobility were
observed for WBH from E2-treated males; however, these bands were absent from
WBH of control males (Figure S2A, lanes 1, 2). Furthermore, the proteins induced
after E2 exposure were successfully purified by the two-step chromatography (Figure
1
S2A, lanes 3, 4, and 5) and stained positively for carbohydrates, phosphorus, and
lipids (Fiure. S2B), confirming that the purified proteins were
phospholipoglycoproteins.
Figure S2. (A) Native-PAGE (4%–7%) of whole body homogenate (WBH) and
fractions of zebrafish WBH. Lane 1, WBH from control male; lane 2, WBH of
E2-treated male; lanes 3 and 4, Vtg fractions from Sephacryl S-300 chromatography;
lane 5, Vtg fractions from DEAE-Sepharose chromatography. (B) Detection of
carbohydrate, lipid, and phosphorus components in zebrafish Vtg. The purified Vtg
was run on native-PAGE and stained with Sudan black (lane 1), Schiff reagent (lane
2), and methyl green (lane 3).
Specificity of the antiserum to zebrafish Vtg
In SDS-PAGE under reducing conditions, purified Vtg showed two major bands
corresponding to approximately 143 and 180 kDa along with three faint bands at
approximately 114, 103 and 96 kDa (Figure S3A). Western blot analysis was
performed to determine the specificity of the antiserum. Anti-Vtg antibody reacted
with purified Vtg and WBH from E2-treated male fish, but not with WBH from
2
control male zebrafish (Figure S3B).
Figure S3. (A) SDS-PAGE (4%–9%). Lane 1, marker; lane 2, purified Vtg. (B)
Western blot. Lane 1, homogenate from E2-treated male zebrafish; lane 2, homogenate
from control male zebrafish; lane 3, purified Vtg.
3
Matrix-assisted laser desorption/ionization-time of flight/time of flight mass
spectrometry (MALDI-TOF/TOF MS) analysis
The two protein bands were identified by MALDI-TOF/TOF-MS (Table S1, Figure
S4-S9). The peptide sequences of band I were highly homologous with Vtg 1, Vtg 2,
Vtg 4, and Vtg 7; the peptide sequences of band II were similar with those of Vtg 2,
Vtg 4, Vtg 5, Vtg 6, and Vtg 7. Further, the matched sequences of Vtg peptides had
two similar sequences (VQVDAILALR, LEFEVQVGPR). Thus, the purified proteins
were confirmed as zebrafish Vtgs.
Table S1. MALDI-TOF/TOF-MS identification of the purified proteins
Band no.
I
II
No. of
mass value
matched
Accession No.
Protein name
Mw (kDa)
MASCOT
score
gi|113678458
vitellogenin 2 isoform 1
181.208
789
14 (13)
gi|166795887
vitellogenin 1
150.308
456
9 (8)
gi|160420306
vitellogenin 4
149.328
393
9 (8)
gi|156713467
vitellogenin 7
149.480
309
8 (7)
gi|160420306
vitellogenin 4
149.434
1121
17 (17)
gi|68448530
vitellogenin 5
149.609
962
15 (15)
gi|303227889
vitellogenin 6
151.677
494
11 (11)
gi|156713467
vitellogenin 7
149.480
381
8 (8)
gi|113678458
vitellogenin 2 isoform 1
181.208
115
3 (2)
4
Figure S4. Matrix-assisted laser desorption/ionization-time of flight/time of flight
mass spectrometry (MALDI-TOF/TOF MS) analysis of bane I and band II
5
Figure S5-1. MALDI-TOF/TOF MS mass spectra of 8 parent mass peaks of band
I in Fig. S4
6
Figure S5-2. MALDI-TOF/TOF MS mass spectra of another 8 parent mass peaks
of band I in Fig. S4
7
Figure S5-3. MALDI-TOF/TOF MS mass spectra of another 8 parent mass peaks
of band I in Fig. S4
8
Figure S5-4. MALDI-TOF/TOF MS mass spectra of 1 parent mass peak of band
I in Fig. S4
9
Figure S6. MALDI-TOF mass spectra of band II
10
Figure S7-1. MALDI-TOF/TOF MS mass spectra of 8 parent mass peaks of band
I in Figure S6
11
Figure S7-2. MALDI-TOF/TOF MS mass spectra of another 8 parent mass peaks
of band I in Figure S6
12
Figure S7-3. MALDI-TOF/TOF MS mass spectra of another 8 parent mass peaks
of band I in Figure S6
13
Figure S7-4. MALDI-TOF/TOF MS mass spectra of another 1 mass peak of
band I in Figure S6
14
Figure S8. Identification of combined MS and MS/MS spectra of bane I by searching
the mass spectrometer data against the NCBInr-zebrafish database via MASCOT tools
15
Figure S9. Identification of combined MS and MS/MS spectra of bane II by searching
the mass spectrometer data against the NCBInr-zebrafish database via MASCOT tools
16
Optimal assay conditions for the sandwich ELISAs
A sandwich ELISA for Vtg detection was developed, using anti-Vtg antibody as
capture antibody, HRP-labeled anti-Vtg IgG as detecting antibodies, and purified Vtg
as the standard. The concentration of anti-Vtg in coating buffer was 5 μg/mL, a
empirical value obtained from the result of Holbech et al. (2001), and incubated with
four serial dilutions of the HRP-labeled antibody. When the dilution of the secondary
antibody was 1:10 000, the curve showed a wide linear work range with high
maximum absorbance of about 3.0 (Figure S10), similar to sandwich ELISAs
developed for Japanese medaka (Oryzias latipes) and fathead minnow (Pimephales
promelas). This dilution of the secondary antibody was thus selected as the optimum
assay condition.
Figure S10. Determine the optimal dilution of HRP-labeled anti-Vtg IgG in the
sandwich ELISA for the quantification of zebrafish Vtg.
17