Biomedical Engineering Reference
In-Depth Information
Put the plate on the cooling plate of a horizontal electrophoresis
apparatus. Fill the buffer chambers with 1:1 diluted Soln. B (
=
elec-
trode buffer) and connect the electrode buffer to the gel by filter
paper bridges, wetted with electrode buffer. The filter paper cov-
ers the gel for about 5 mm. Remove carefully air bubbles between
papers and gel.
Remove the comb and fill the samples, 5 - 10
µ
l each, into the
pockets. It is recommended to cover the gel with a glass plate being
on the filter bridge.
Cool the gel to about 10
◦
C,andthenruntheelectrophoresis
with cv of 8 - 15 V
/
cm.Proteinsaswellasbromophenolbluemove
to the anode (+).
In the case of protein ligands the detection of ligates and
Capillary blotting
ligand-ligate complexes is done by blotting. After finishing the elec-
trophoresis, remove the upper glass plate and the buffer bridges and
cover the gel with a dry blotting membrane of same size (remove
air bubbles).
The membrane is covered with a 3 to 4 mm stack of dry filter
paper. Finish the sandwich with a glass plate and wrap all with
plastic foil. After incubation overnight in a refrigerator remove the
membrane, wash with PBS and block with an appropriate buffer,
e.g., 0.5% BSA in PBS, 30 min.
Identify the ligate by specific antibodies as described in Proto-
col 2.5.4 (Western blot).
References
Heegard HH, Bjerrum OJ (1991) Anal Biochem 195:319
Takeo K (1987) In: Chrambach A, Dunn MJ, Radola BJ (eds.) Advances in
electrophoresis, vol. 1, pp 229-279, VCH, Weinheim
Taketa K (1991) J Chromatogr 569:229
2.1.11 Two-Dimensional Polyacrylamide Gel Electrophoresis
(2D-PAGE; IEF followed by SDS-PAGE)
Generally, two-dimensional gel electrophoresis (2D electrophore-
sis) is the sequential separation of a protein mixture in an electrical
field, whereas the conditions of the first separation are different
from those of the second one, running rectangular to the first. First
and second dimension may differ in acrylamide concentration, pH,
buffer composition, by addition of detergents or urea, or type of
carrier (polyacrylamide or agarose). Mostly the combination of iso-
electric focusing (IEF) for the first separation and SDS-PAGE for
the second is described as “2D-PAGE.” When using soluble carrier
ampholytesinIEFforthefirstdimension,themethodisdesignated
as O'Farrell technique; it is named IPG-Dalt when ampholytes are
co-polymerized (immobilized) within the polyacrylamide matrix
(Görg 1988).
