Soybean Proteomics - Plant Proteomics: Methods and Protocols - page 307

containing 8 M urea, 2 % Nonidet P-40, 2 % ampholine (pH 3.5-10),
5 % 2-mercaptoethanol, and 5 % Polyvinylpyrrolidone (PVP)-40;
B-buffer [ 13 ] containing 7 M urea, 0.2 M thiourea, 0.2 mM tribu-
tylphosphine (TBP), 0.4 % CHAPS, 5 % PVP-40, and 2 % ampho-
line (pH 3-10); and C-buffer containing 8.5 M urea, 2.5 M
thiourea, 5 % CHAPS, 1 % dithiothreitol (DTT), 1 % Triton X-100,
and 0.5 % ampholines (pH 3-10 and 5-8) were tested. Combination
of the phenol-based method with C-solubilization buffer gener-
ated high quality proteome maps in terms of well-separated
resolved spots, spot intensity, and the number of proteins in the
2-DE gels with no horizontal streaking and high background noise
levels. Authors claim that this optimized protein extraction proto-
col is equally effi cient for all other tissues and organs of soybean,
including the hypocotyl, leaf, petiole, stem, fl ower bud, and fl ower.
Soybean root proteins extracted in 10 % TCA and 0.07 %
2-mercaptoethanol in acetone followed by subsequent solubiliza-
tion in the lysis buffer containing 8 M urea, 2 M thiourea, 5 %
CHAPS, and 2 mM TBP results high quality gel with a good num-
ber of resolved protein spots [ 15 , 16 ]. Addition of DTT and PVP
in the soybean protein extraction buffer was found to be effective
in enhancing the number of resolved spots in gels [ 17 , 18 ]. Xu
et al. [ 19 ] and Natarajan et al. [ 20 ] used a modifi ed lysis buffer
devoid of thiourea and TBP, that comprises of 9 M urea, 1 %
CHAPS, 1 % DTT and 1 % ampholytes (pH 3-10), to solubilize
the TCA/acetone precipitated proteins of soybean leaf and mature
seed, respectively (Table 2 ).
Recently, Barbosa et al. [ 21 ] have analyzed mature seed pro-
teome by extracting proteins in 50 mM Tris-HCl (pH 8.8), 1.5 mM
KCl, 10 mM DTT, 1.0 mM phenylmethylsulfonyl fl uoride (PMSF),
and 0.1 % SDS followed by precipitation in 0.1 M ammonium ace-
tate in methanol. However, phenol based protein extraction was
shown to be more effective in extracting seed proteins both at
mature [ 22 ] and seed fi lling stages [ 23 ]. In contrast to TCA/ace-
tone or Tris-HCl buffer, protein extracted in buffer comprises of
50 % phenol, 0.45 M sucrose, 5 mM EDTA, 0.2 % 2- mercaptoethanol,
50 mM Tris-HCl (pH 8.8) when solubilized in 8 M urea, 2 M
thiourea, 4 % CHAPS, 2 % Triton X-100, 50 mM DTT, ampho-
lytes produced large number of reproducible protein spots
(Table 2 ). Mooney and Thelen [ 22 ] compared the quality and
purity of the acetone-precipitated and phenol extracted proteins.
Occurrence of minimal horizontal streaking in 2-DE gels for
phenol-extracted proteins indicates that acetone-precipitated sam-
ple contains more non-protein molecules. Ahsan et al. [ 24 ] evalu-
ated a modifi ed phenol method for extraction and analysis of
soybean leaf proteome in response to ozone stress (Table 2 ). The
leaf tissue was fi rst homogenized in extraction buffer containing
0.5 M Tris-HCl (pH 8.3), 2 % Nonidet P-40, 20 mM MgCl 2 , 2 %
2-mercaptoethanol, 1 mM PMSF and 0.7 M sucrose. An equal