Protein Protocols Protein Protocols

Semidry Protein Blotting 323
2. Membranes and filter papers: PVDF (0.2 µm, 200 × 200 mm, Bio-Rad) or nitrocellulose
(0.45 µm, Schleicher & Shuell). Filter papers are chromatography papers (grade 3 mm
CHr, Whatman). Thicker blotting papers can also be used (Pharmacia-LKB, filter paper
for blotting, 200 × 250 mm).
3. Electroblotting apparatus: Proteins are transferred with a Trans-Blot SD semidry cell
(Bio-Rad). The anode of the apparatus is made of platinum-coated titanium and the
cathode is made of stainless steel. The maximum gel size that can be used with this
apparatus is 25 cm × 18.5 cm.
4. Coomasie staining: Proteins in the 2-D gel and on the blot (Fig. 1) are stained with a
0.025% (w/v) solution of Coomasie Brilliant Blue R250 solubilized in 43% (v/v)
methanol and 7% (v/v) acetic acid. The destaining solution contains 30% (v/v) methanol
and 7% (v/v) acetic acid.
All chemicals and methanol are of analytical reagent grade. Metallic contaminants in lowgrade
methanol normally deposit on the electrodes.
3. Method
Note: To avoid membrane contamination, always use forceps or wear gloves when
handling membranes.
3.1. Preparation for Semidry Blotting
1. Prepare the transfer buffer the day preceding the blotting experiments and store it at 4°C.
2. After the separation of proteins by sodium dodecyl sulfate (SDS)-PAGE or 2-D PAGE,
the gel is briefly rinsed in distilled water for a few seconds and then equilibrated in
transfer buffer for 10 min under gentle agitation (see Note 2).
3. During the equilibration, the filter paper and the transfer membrane are cut to the dimensions
of the gel. Six pieces of filter paper per gel are needed for each gel–membrane sandwich
(or two pieces of thick filter papers).
4. If the hydrophobic PVDF membrane is used, it should be prewetted prior to equilibration
in transfer buffer. Immerse the membrane in a small volume of 100% methanol for a few
seconds, until the entire membrane is translucent, rinse it in deionized water and then
equilibrate it in transfer buffer for 3–5 min. It is important to keep the membrane wet at all
times since proteins will not bind to the dried PVDF membrane (see Note 3).
For hydrophilic nitrocellulose membrane, wet it in transfer buffer directly and allow it to
soak for approx 5 min (see Notes 4 and 5 and Table 2 for the description of the different
transfer membranes).
5. If multiple full-size gels are to be transferred at one time, there is a necessity to interleave
a dialysis membrane between the gel-membrane pairs to prevent proteins being driven
through membranes into subsequent stacks.
Cut a piece of dialysis membrane with the appropriate molecular weight cutoff to the
dimensions of the gel and soak it in the transfer buffer (see Note 6).
3.2. Assembly of the Semidry Unit
The assembly of a semidry electroblotting apparatus is represented in Fig. 2. Four
mini gels can also be transferred at the same time by placing them side-by-side on the
anode platform.
1. The filter papers are briefly soaked in transfer buffer for a few seconds.
Place a prewetted filter paper onto the anode. Use a pipette or a painter roller to eliminate
all air bubbles by pressing firmly all over the area of the paper (see Note 7). If a thin filter
paper is used, add two more sheets and remove air bubbles between each layer.