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Western Blot
时最常用的两种膜是硝酸纤维素膜（
nitrocellulose
，
NC
膜）和
PVDF
膜（又称
Positively charged nylon
）
（
Polyvinylidene fluoride
，聚
偏
二氟乙烯膜）。
这两种膜各自有什
么特点？我们的实验中该选择哪一种呢？来看下面的文字。
（摘自《
Making and using Antibodies
》）
A
study of the performance of nitrocellulose, mixed
ester, nylon, and
covalent-binding PVDF
memberanes after passive protein adsorption
an
d also after electrotransfer was done
with several different proteins
labeled
with
125
Iodine. The
membranes exhibited different binding
cap
acities in passive adsorption tests
with labeled bovine serum albumin.
The
PVDF showed the least, and the regenerated
cellulose and nylon m
embranes showed
the most protein binding. Nitrocellulose and mixed
es
ter membranes were midway between. In
tests measuring protein retenti
on, PVDF
retained the most bound protein when washed with
detergents
or 5% skimmed milk. All the
membranes showed virtually the same
bindi
ng capacity as measured by
autoradiography when tested under
electrot
ransfer conditions with
Towbin's buffer. In passive adsorption
tests,
the membranes wxhibited a broad
range of capacities but gave similar
results in electrotransfer tests. These
differences were ascribed to
active
migration of protein into the membrane matrix
instead of simp
le diffusion and the
increased hydrophobicity of Towbin's transfer
bu
ffer because of the inclusion of
methanol.
上面这段文字指出在被动扩散转移蛋白时，
几种膜之间结合蛋白的能力差别明显；
但是
当使用转膜仪转移
蛋白时，各种膜之间的差别就很小了
。
The choice of membrane used for Western
Blot is more critical if the
blotted
protein must maintain its native conformation for
detection b
y the antibody. For example,
a comparison using a guanosine
triphosph
ate(GTP)-overlay assay showed
that the activity of a bovine GTP-
bindi
ng protein was barely detectable
after transfer to hydrophobic PVDF
m
embranes but was clearly detected
after transfer to nitrocellulose.
W
estern blot analysis showed the GTP-
binding protein to be present on
both
PVDF and nitrocellulose membranes, with slightly
more detected o
n the PVDF membranes.
The authors speculated that the poor
performanc
e of PVDF in the GTP-overlay
assay may have been due to an inability
of GTP-binding protein, thus
immobilized, to renature correctly.
Ther
efore, nitrocellulose might be
preferred for a Western blot procedure,
in which detection requires that the transferred
protein regain its
native conformation
after transfer, such as when the blotting agent
r
ecognized three dimensional structure;
for example, an antigenic epit
ope
consisting of noncontiguous residues.
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