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Fig. 5. Western blot analysis of V-ATPase expression in subcellular fractions of
encysted embryos, employing three polyclonal antibodies raised against (A,B)
the native V1 complex, (C,D) recombinant d-subunit of the
V0 domain or (E) recombinant a-subunit of the V0 domain
from Manduca sexta larvae. A biotinylated secondary antibody was used
for blots in A, C, D and E. An alkaline phosphatase-conjugated secondary
antibody was used for blot in B to allow visualization of subunit A (70 kDa),
which is obscured in biotinylated blots by a strong non-specific band (ns)
visible in blots A, C, D and E. Subcellular fractions from 0 and 8 h of
development were run side by side (H, heavy membrane; V140,
microsomal vesicles pelleted at 140 000 g; S140,
post-ribosomal supernatant from 140 000 g spin; W,
V140 pellet resuspended in buffer of low ionic strength and
pelleted a second time; Ms, Manduca sexta heavy membrane
preparation). All immunoreactive bands were identified by comparison with
control blots incubated with secondary antibody only. Subunit designation (in
parentheses) and apparent Mr (in kDa) for each band are
listed to the left of each blot.
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