Biomedical Engineering Reference
In-Depth Information
(6) 1.3 M stock dithiothreitol (DTT) solution: 2 g DTT is
dissolved in Milli-Q 18
water and volume adjusted
20
◦
C. This solution should not be
−
to 10 ml, store at
heated.
(7) Stock ampholines/pharmalyte solution: Equal volumes
of pH 3.5-10 ampholines and pH 3-10 pharmalyte are
mixed prior to use, store at 4
◦
C for short periods. These
broad pH range IPG buffers can be replaced with narrow
range buffers depending on the first dimension pH range.
(8) Stock 0.2% (w/v) bromophenol blue solution: 20 mg of
bromophenol blue is diluted in 10 ml with distilled water.
The solution is then filtered and stored at room tempera-
ture.
(9) 14% PAGE solution: 46.5 ml 30% acrylamide-bis solu-
tion, 26.9 ml Milli-Q 18
water (or equivalent), 25 ml
1.5 M Tris-HCL, pH 8.8, 1 ml 10% SDS, 35
l
TEMED (
N
,
N
,
N
,
N
-tetramethylethylenediamine) and
0.5 ml 10% APS and gently agitated under mild vacuum
for 1 h. The TEMED and APS are added immediately
prior to pouring the gel solution into the casting unit.
These volumes are sufficient for one 24 cm gel. 10-15%
solutions may be used for second dimension separation
depending on the mass range separation required.
(10) Bind silane solution: 16
μ
μ
l Plus One Bind Silane, 400
μ
l
glacial acetic acid, 16 ml ethanol, 3.6 ml Milli-Q 18
water. Sufficient for 12 24 cm
×
20 cm plates, cannot be
stored.
(11) Equilibration buffer: 6 M urea, 30% (v/v) glycerol,
50 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, store at
20
◦
C
−
for 4-6 weeks.
(12) Agarose overlay 0.5% (w/v): melt 1 g of agarose in 200 ml
of 1X SDS electrophoresis buffer in a microwave on low
heat. Great care must be taken using a microwave and
solution must be allowed to cool to luke warm before
moving. Add bromophenol blue solution for a pale blue
colour.
(13) Tris glycine-SDS electrophoresis running buffer: 0.025 M
Tris Base, 0.192 M glycine and 0.1% sodium dodecyl sul-
phate.
(14) Gel fixing solution: 35% (v/v) methanol and 7.5% (v/v)
acetic acid made to required volume with Milli-Q 18
water, store at 4
◦
C.
(15) Acetonitrile wash
solution:
50%/50% acetonitrile
and Milli-Q 18
water
(v/v),
store
at
room
temperature.
Search WWH ::
Custom Search