Biomedical Engineering Reference
In-Depth Information
After finishing the 2D-PAGE, proteins are separated both by
their isoelectric points (pI) and molar masses (M
r
).
The main advantage of the IPG-Dalt protocol is a much higher
reproducibility in gel preparation and running conditions, and
mostly higher resolution of the sample.
2.1.11.1 First Dimension: Isoelectric Focusing (IEF)
Preparation of Immobiline Gels
A
29.1% acrylamide (w/v), 0.9% N,N
-methylene bisacrylamide
Solutions/Reagents
(w/v) (30% T, 3% C), stored with 10% humid, neutral mixed-
bed ion exchanger (w/v) in ddH
2
O
B
10% (v/v) TEMED in ddH
2
O
C
10% (w/v) ammonium persulfate in ddH
2
O (prepare freshly)
rehydration solution
10
:8M urea, 1% CHAPS (w/v), 15 mM
DTT, 0.2% ampholyte pH 3 - 10 (v/v)
D
Volumes for a IPG (IEF) gel 180
×
180
×
0.5 mm (glass plate 200
×
200 mm; total volume 16.2 ml) are given in Table 2.10.
Give some drops of water onto a clean 200
200 mm glass plate.
Place a piece of GelBond foil, hydrophilic side upward, on the plate
(it is recommended to draw a thin line with a pencil on one side
of the foil for easier identification of, for example, the acidic side).
×
Table 2.10.
Casting protocol for Immobiline gels with linear pH gradient
µ
Solution (
l)
pH 4 - 9
pH 4 - 12
%T=4,%C=3
%T=5,%C=3
Acidic
Basic
Acidic
Basic
Immobiline pK 3.6 448 79 513 -
Immobiline pK 4.6 127 229 190 40
Immobiline pK 6.2 125 194 172 111
Immobiline pK 7.0 12 160 159 56
Immobiline pK 8.5 135 38 26 282
Immobiline pK 9.3 119 358 28 118
Immobiline pK 10.0 - - 22 176
Immobiline pK
>
13 - - - 287
Solution A 1080 1080 1350 1350
Glycerol (g) 2 - 2 -
H
2
O 4396 5908 3910 5584
Mix, adjust pH 7.0 with 4 N NaOH and 4 N acetic acid, respectively
Solution B
54
54
54
54
Solution C
32.4
32.4
32.4
32.4
Data according to Görg (2003)
10
24 g urea and 0.5 g mixed-bed ion exchange resin are filled up with H
2
O
to 50 ml.Themixtureisstirredfor10min at RT, than the resin is filtered
off. CHAPS, DTT, and ampholyte are added in the indicated amount to
the clear urea solution.
