Manual

5.2.2. Strongly Chaotropic 2-D Rehydration/Sample
Buffer
(7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 0.2%
Bio-Lyte 3/10 ampholyte, 0.002% Bromophenol Blue).
Component Final Concentration Amount to make 2 ml
Urea (FW 60.06) 7 M 0.84 g
Thiourea (FW 76.12) 2 M 0.304 g
CHAPS
*
4% (w/v) 0.08 g
DTT (FW 154.3) 50 mM 15.4 mg
100X Bio-Lyte 0.2% (w/v) 20 µl
3/10 ampholyte
**
Bromophenol Blue 0.002% (w/v) 4 µl of a 1%
(w/v) solution
Proteomic grade 1.1 ml
water
*
Other neutral or zwitterionic detergents can also be used at concentrations
of 1% to 2% (w/v) to improve solubilization of membrane and hydrophobic
proteins. Examples are n-octyl-β-D-glucopyranoside, SB3-10 (N-decyl-N,N-
dimethyl-3-ammonio-1-propanesulfonate) and ASB14 (tetradecanoylamido-
propyl-dimethylammonio-propane-sulfonate).
**
Use an ampholyte buffer that corresponds to the pH range of the IEF
separation to be performed. For example, ReadyStrip
™
micro-range buffers
with ReadyStrip micro-range IPG strips and ReadyStrip 7-10 buffer with
ReadyStrip pH 7-10 IPG strips. Bio-Lyte 3/10 ampholyte can be used with
all other ReadyStrip IPG strip pH ranges.
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