Protein Protocols Protein Protocols

322 Gravel
Fig. 1. Plasma proteins separated by two-dimensional polyacrylamide gel electrophoresis
and (A) stained with Coomasie Brilliant Blue R250 or (B) transferred to PVDF membrane
using the semidry system (2 h, 15 V) with Towbin buffer diluted 1:2 in water and stained with
Coomasie Blue.
and then scanned with IR-MALDI-MS (infrared matrix-assisted laser desorption/ ionizationmass
spectrometer). They demonstrated that the sensitivity for protein detection
was comparable if not better than that of sensitive silver-stained gels (3).
The blot analysis generally requires small amounts of reagents, the transferred
proteins on membrane can be stored for many weeks prior to their use and the same
blot can be used for multiple successive analyses. For reviews on the basic principles
involved in performing protein blotting and for an overview of some possible applications,
see articles by Garfin et al. (1), Beisiegel (4), Gershoni et al. (5), Towbin et al. (6), and
Wilkins et al. (7).
In the following sections, we describe a protocol for semidry blotting that uses a
simple buffer system (8). The efficacy of this method is illustrated in Fig. 1 with
human plasma proteins separated by two-dimensional polyacrylamide gel electrophoresis
(2-D PAGE), transferred on PVDF membrane and stained with Coomasie Blue. The
blot pattern is compared to the Coomasie Blue staining of the same protein sample
before transfer from 2-D PAGE. The resolution, shape, and abundance of protein spots
on membrane are comparable to the 2-D polyacrylamide gel pattern. This blotting
procedure allows a good and almost complete elution of proteins from the gel and their
immobilization on the membrane.
We also review in Subheading 4. the principal types of transfer matrix and the
different discontinuous or continuous buffers that can be used with this technique.
2. Materials
1. Buffer: The transfer and equilibration buffer is the Towbin buffer diluted 1:2 with distilled
water: 12.5 mM Tris, 96 mM glycine, and 10% (v/v) methanol (9) (see Note 1 and Table 1).