Manual

3. For each gram of dry reagent, add 1.1 ml of PSB
diluent.
4. Vortex periodically and incubate at room temperature
until you have a clear solution (2–3 min).
5. Add reducing agents, protease inhibitors, and carrier
ampholyte as needed (Table 2).
Table 2. Additions to PSB solution recommended
for various applications. Note that though the
applications listed often require use of chaotropes and
detergents, these agents are already included in the PSB
solution.
Component Protein
Extraction IEF Separation
MicroRotofor Cell IPG Strip
Carrier ampholyte NA 2% (w/v) 0.2% (w/v)
DTT* 50–100 mM 50–100 mM 50–100 mM
or
TBP* 2–5 mM 2–5 mM 2–5 mM
Protease inhibitor According to NA NA
manufacturer
Bromophenol Blue NA 0.002% (w/v) NA
Glycerol NA 10% NA
*Not needed if reduction-alkylation is performed at step 17.
6. Suspend the wet bacterial cell pellet (~50 µl) in 500 µl
of bacteria suspension buffer.
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