Biomedical Engineering Reference
In-Depth Information
The electrophoresis is run at 1000 V cv for 30 - 45 min. After finish-
ing, the cellulose sheet is dried in a stream of warm air. For visual
detection of amino acids the cellulose sheet is sprayed with Soln. B
and incubated in a drying oven at 110 - 120
◦
C until blue spots
appear. Autoradiography or a PhosphoImager detects
32
P-labled
amino acids.
Phosphorylated amino acids move in the order serine phos-
phate
>
threonine phosphate
>
tyrosine phosphate.
For quantitative determination the corresponding spots and
a blank area of same size are cut out. The cellulose is burned with
concentrated nitric acid and the residue is used for phosphate de-
termination according to Protocol 1.3.2, enzymatically by coupled
optical test and in the case of
32
P-labeling by counting radioactivity.
References
Hunter T, Sefton BM (1980) Proc Natl Acad Sci USA 77:1311
2.3 Aid in Electrophoresis
A lot of companies offer numerous kits for calibration and staining
of electropherograms. These kits simplify working with gels, but
though they are optimized with respect to their composition, they
are mostly expensive and self-made reagents give the same, and
in some cases better, results. Besides, the following protocols gives
a better understanding of mechanisms of calibration and staining.
2.3.1 Marker Dyes for Monitoring Electrophoresis
2.3.1.1 Anodic Systems
0.02 g bromophenol blue in 0.25 ml ethanol and 0.02 g Py-
Solutions/Reagents
ronin Y (Pyronin G) in 0.25 ml ddH
2
O separately dissolved,
combined and supplemented with 0.5 ml glycerol or 1 ml 50%
sucrose (w/v) in ddH
2
O, filled up with ddH
2
Oto2ml
µ
µ
0.05
l sample solution of
all SDS-PAGE systems with a pH of separation gel
>
5. The color
of bromophenol blue solution switches from yellow-brown to blue-
violet. Better than bromophenol blue, especially at separation of
small polypeptides, is Orange G (C.I. 16230), because it moves
directly with the electrophoresis front. Unfortunately, Orange G is
not as easily visible as bromophenol blue or Pyronin Y.
l of this dye solution are added per 1
2.3.1.2 Cathodic Systems
0.5% basic fuchsin (w/v), 50% sucrose (w/v) in ddH
2
O
Solutions/Reagents
µ
0.05
l of this solution are added per microliter sample. Further
addition of sucrose or glycerol is not necessary.
