Bio-Rad ReadyPrep™ Reduction-Alkylation Kit - Manual (Page 4)

Section 1
Introduction
The ReadyPrep reduction-alkylation kit offers an easy and
effective way of improving spot resolution during 2-D
electrophoresis. This general-purpose kit minimizes streak-
ing and aberrant spot migration by reducing and irreversibly
blocking the formation of inter- and intramolecular disulfide
bonds prior to isoelectric focusing (IEF). Disulfide bond
formation is particularly problematic for basic proteins
because of the increased rate of their formation in an
alkaline environment. Moreover, many reducing agents
become negatively charged during isoelectric focusing and
migrate off the IPG strip, thus allowing disulfide bonds to
re-form (Görg 2000, Hoving 2002). The ReadyPrep
reduction-alkylation kit eliminates the potential for disulfide
bond formation during IEF by first reducing the disulfide
bonds with tributylphosphine (TBP), a powerful nonionic
reducing agent, and then alkylating the sulfhydryl groups
with iodoacetamide (Herbert et al. 2001). This treatment
produces a spot pattern with more spots, fewer streaks,
and greater reproducibility. The entire process can be
completed directly in the rehydration/sample buffer in <2 hr
with very little hands-on time. This kit is compatible with
other ReadyPrep sample preparation kits.
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