Biology Reference
In-Depth Information
3. Western blotting. Denature 20
g of cytoplasmic extracts for
5 min at 98 °C in 1× SDS sample buffer and load on SDS-
PAGE gel (10 × 8 cm) in Tris-glycine Buffer. Use a biotinylated
protein ladder (size range of 9-200 kDa) as standard to deter-
mine molecular weight. The electrotransfer to 20
μ
m nitrocel-
lulose membrane is performed overnight at 360 mA and 4 °C
in electrotransfer buffer. The membrane is prestained in
Ponceau S Solution to verify the transfer, washed with 25 ml
TBS for 10 min at room temperature and incubated in 25 ml of
blocking buffer for 2 h at room temperature. The membranes
are washed three times for 5 min each with 25 ml of TBS/T
and incubated with primary rabbit monoclonal antibody
(1:1,000) in 15 ml primary antibody dilution buffer with gentle
agitation overnight at 4 °C. The day after, the membrane are
washed three times for 5 min each with 20 ml of TBS/T and
incubated in 15 ml of blocking buffer, in gentle agitation for
2 h at room temperature, with an appropriate HRP-conjugated
secondary antibody (1:2,000) and an HRP-conjugated anti-
biotin antibody (1:1,000) to detect biotinylated protein
marker. Finally, after three washes each with 20 ml of TBS/T
for 5 min, the membranes are incubated with chemolumines-
cent substrate according to the manufacturers instruction and
exposed to X-ray fi lm. X-ray fi lms for chemiluminescent blots
are analyzed by Gel Doc 2000 using Quantity One program to
elaborate the intensity data of your specifi c protein targets.
Ponceau S staining can be used as normalization control, but
others marker proteins may be taken as reference too and are
specifi cally reported. The data of Western blot assay (Fig.
4b
)
show a clear increment of p27
Kip1
protein expression in the sam-
ple from cells treated with Rpep-PNA-a221.
μ
4
Notes
1. FACS analyses, such as those shown in Fig.
1
(panels A-C)
even performed several times and obtaining consistent results,
are compatible with uptake of Fl-Rpep-PNA-a221 by target
cells, but cannot exclude the possibility that the fl uorescence
signal is due at least partially to cell-surface interactions, caused
by the positive charged polyarginine peptide, which might
interact strongly with negative charged protein components.
Therefore FACS-based assays should be combined with
microscope-assisted analyses (
see
Fig.
1
, panels D-F) to con-
fi rm that PNAs are internalized within target cells. Confocal
analyses are in addition necessary if compartmentalization
within intracellular compartments should be determined.
2. In order to determine the concentrations of Fl-Rpep,
Fl-PNA-a221, and Fl-Rpep-PNA-a221 to be employed for in
