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3.233.9. SDS-PAGE analysis
Protein analysis was performed on 12% SDS-PAGE as descrihcd hy Larmmli
(1970). Protein samples were reduced by boiling for 5-10 niin. in loading huffer
containing 570 70-mercnptoethanol and then centrifuged at IO.DW g for
5
min. prior to
loading the gel. Electrophoretic anulyses were performed using a hlighty Small Unit
11
system (model S E 250; Pharmacia Biotech Asia Pacific Ltd. Hongkong). Vertical
,lab gels, containing 12% (w/v) resolving gel and 5 6 stacking gel concentrations of
:icrylamide, were run at a constant current of 15 mA for 6 h. The proteins on PAGE
gels were fixed in 4570 ~nethanol and 10% acetic acid in distilled water, stained in
0.25% Coomassie brilliant blue R-250 dissolved in 1070 acetic acid and 50%
methanol in water. The gels were destained for 3 h in 5 6 methanol and 7% acetic
acid in distilled water and documented using photo gel documentation system (Vilber
Lourmat, France).
3.2.3.3.10. Western blotting
After SDS-PAGE, gels were soaked in the tranafer huffer (25
rnM
Tris. 192
mM glycine, 30% methanol, pH 8.4) and electmtransferred at 0.8 r n ~ . c m " for 50
min. with a semi-dry blotting apparatus, Semiphor Transphor Unit (Pharmacia
Biotech Asia Pacific Ltd., Hongkong) onto a 0.45 Km nitrocellulose membrane
(Advantech
MFS
Inc., CA, U.S.A) as described by Towbin
er al..
(1979). The
Western blot was saturated in Tris NaCl buffer (20
mM
Tris HCI, pH 8.0. 150
mM
NaCI) with 3 % gelatin and incubated for 3 h with a 1:1000 dilution of rabbit
antiserum against barley chitinase o r cah in the same buffer but with I % gelatin. The
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