Bio-Rad PROTEAN® IEF Cell Accessories User Manual
Page 35
1.
First Dimension
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8-9.8 M Urea*
4.8-5.8g/10ml
8-9.8 M Urea*
4.8-5.8 g/10 ml
0.5% CHAPS
50 mg/10ml
1-4% CHAPS*
100-400 mg/10 ml
10 mM DTT
15 mg/10ml
15-100 mM DTT*
23-150 mg/10 ml
(Dithiothreitol)
(Dithiothreitol)
or,
or,
2mM Tributylphosphine**
2mM Tributylphosphine**
0-0.2% (w/v) Bio-Lytes
†
0-0.2% (w/v) Bio-Lytes
†
0.001% Orange G
0.001% Orange G
or Bromophenol Blue
or Bromophenol Blue
* The amounts of Urea, CHAPS, DTT, and Bio-Lytes required depend on the sample solubility. The amounts listed here serve
as a general guideline. The optimal rehydration buffer composition for each sample type is best determined empirically.
** Tributylphosphine is an alternative reducing agent for DTT.
4,5,6,7
†
pH gradient corresponding to IPG Strip gradient.
IPG Strip Rehydration
7
7
7
7 c
c
c
cm
m
m
m R
R
R
Re
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a
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ad
d
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dyy
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Sttttrrrriiiip
p
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1
1
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17
7
7
7 c
c
c
cm
m
m
m R
R
R
Re
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e
ea
a
a
ad
d
d
dyy
y
yS
S
S
Sttttrrrriiiip
p
p
p
Minimum rehydration volume
125 µl
300 µl
Rehydrates strip to a gel
thickness of 0.5 mm, and gel
composition of 4%T/3%C
Maximum rehydration volume
250 µl
600 µl
Increased rehydration volume
allows for larger protein sample
load, facilitates entry of larger
proteins, and minimizes protein
solubility problems
Sample Loading Following ReadyStrip Rehydration
Samples can be applied following rehydration immediately prior to focusing. The
rehydrated ReadyStrips are positioned in the focusing tray and covered with mineral oil or
similar overlay material. The sample is then added to the sample loading wells on the
channel in the focusing tray. The recommended sample volume for tray loading is
up to 30 µl/ReadyStrip.
32