Protein Protocols Protein Protocols

Semidry Protein Blotting 331
Table 3 (continued)
Blocking Agents for Blots
Blocking agent Remarks
Other:
Polyvinylpyrolidone As adjunct to Tween-20, PVP-40 produces
(PVP-40, 40,000 M r) backgrounds lower than those of Tween-20
alone, approximately doubling the signalto-background
ratio (without decreasing
specific immunoreactivity in Western
blots) Should be used at a concentration of
1% (w/v) and added to 0.05% (w/v)
Tween-20 (27).
Towbin buffer diluted 1:2 with water and Laurière, buffer) for transferring plasma proteins
from 2-D PAGE by semidry method and we similarly found no advantage in
using discontinuous buffer systems (unpublished results).
2. Rinsing and equilibrating the gel facilitate the removal of electrophoresis buffer salt and
excess of detergent. If salts are still present, the conductivity of the transfer buffer increases
and excessive heat is generated during the transfer. Also, an equilibration period
allows the gel to adjust to its final size prior to electroblotting because the gel shrinks in
methanol-containing transfer buffer.
The duration of equilibration depends on the gel thickness. For a 1.5 mm thick gel, 10 min
of equilibration are enough.
Recently, poly(ethylene glycol) polymers (PEG 1000–2000) have been used to complete
electroblotting, in order to obtain better resolution and enhancement of sensitivity of proteins
on membrane. PEG reduces background, raises signal-to-noise ratio and sharpens
protein band (20). After polyacrylamide gel electrophoresis, 30% (w/v) PEG (solubilized
in transfer buffer: 12.5 mM Tris, 96 mM glycine and 15% [v/v] methanol) is applied to
reversibly fix proteins within the gel. The intragel proteins can then be electroblotted
directly onto membranes using the same transfer buffer containing PEG.
3. If the PVDF membrane does dry out during use it can be rewet in methanol. Membranes
that contain adsorbed proteins and that have been allowed to dry can also be rewet in
methanol. In our experience, we have not seen any difficulty in protein staining, glycoprotein
detection and immunostaining after this rewetting procedure in methanol.
4. Nitrocellulose membrane was the first matrix used in electroblotting (10) and is still the
support used for most protein blotting experiments. It has a high binding capacity, the
nonspecific protein binding sites are easily and rapidly blocked, allowing low background
staining and it is not expensive. For blotting with mixtures of proteins, standard nitrocellulose
with a pore size of 0.45 µm should be used first. However, membranes having
0.1 µm and 0.2 µm pore sizes should be tried if some low molecular weight proteins do
not bind efficiently to the standard membrane (1). After staining, the nitrocellulose membranes
become transparent simply by impregnating the membrane with concentrated Triton
X-114 (21). The blot can thus be photographed by transillumination or scanned with a
densitometer for quantitative analysis. Long-term stability (several months) of transparent
stained blots is possible if they are stored at –20°C.