Biomedical Engineering Reference
In-Depth Information
/
was. Continue electrophoresis with 10 - 15 V
cmuntil bromophenol
blue arrives at the anodic paper bridge.
Identify the protein spots as usual, i.e., by staining, autoradio-
graphy, gel overlay, or Western blot. In the latter case the gel must
Western blot
of 2D gel
be separated from the GelBond foil prior to electrotransfer. For
this purpose a film remover (Görg 2003, Fig. 19) is used: The gel
is placed on the cylindrical remover with foil down, clamped on
an edge, and a thin stainless steel or nylon wire is pulled between
foil and gel towards to your body. Cover the gel with the wetted
blotting membrane (cf. Protocol 2.4.3) and transfer membrane as
well as gel to the blotting apparatus.
References
O'Farrel PH (1975) J Biol Chem 250:4007
Dunn MJ, Burghes AHM (1983) Electrophoresis 4:97; 4:173
Görg A, Postel W, Günther S (1988) Electrophoresis 9:531
Righetti PG (1990) Immobilized pH gradients: theory and methodology.
Laboratory techniques in biochemistry and molecular biology, vol. 20,
Elsevier, Amsterdam
Westermeier R (1997) Electrophoresis in practice: a guide to methods and
applications, 2nd ed. VCH, Weinheim
GörgA,ObermaierC,BoguthG,HarderA,ScheibeB,WildgruberR,Weiss
W (2000) Electrophoresis 21:1037
Görg A (2003) Two-dimensional electrophoresis with immobilized pH gra-
dients for proteome analysis. http://www.weihenstephan.de/blm/deg
2.2 Agarose and Paper Electrophoresis
2.2.1 Non-denaturating Nucleic Acid Electrophoresis
This system separates double-stranded DNA (dsDNA) fragments
with a length of 70
80000basepairs(bp)ingelsof3%to0.1%
agarose. For analysis of smaller-fragments (6000 to 1000 bp)PAGE
systems are described in literature with 20 - 3% polyacrylamid.
During sample preparation and electrophoresis DNA will not
be denaturated, i.e., the double strand and higher elements of struc-
ture stay unaffected. To avoid unwanted further fragmentation all
manipulations have to be done without possible contaminations
by fingerprints, droplets of saliva or microorganisms. Sterilized
materials are recommended.
Since protocols for electrophoresis used in DNA sequence anal-
ysis are given, for example, by Sambrock et al., these specialized
methods are not outlined in this chapter.
×
200 mM Tris-acetate, 10 mM EDTA, pH 8.2 (TAE buffer
12
)
A
Solutions/Reagents
B
0.1 - 3.0% agarose (low endoosmosis) (w/v) in 1/5 buffer A
12
Fiftyfold stock of TAE buffer see Chap. 7.4
