Bio-Rad ReadyPrep Reduction-Alkylation Kit User Manual

ReadyPrep

™

Reduction-Alkylation Kit

Instruction Manual

Catalog #163-2090

For technical service, call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD (1-800-424-6723)

Bio-Rad Laboratories, Inc.

2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
[phone]

1-800-424-6723

4110063 Rev A

Table of Contents

Section 1 Introduction……………………………………...1

Section 2 Kit Specifications…………………………......2–3

Section 3 Storage Conditions………………………..........3

Section 4 Instructions for Use………………….............4–6

Section 5 Appendix……………..……………..............7–11

Section 6 References…………..……………..................12

Section 7 Product Information………………….........13–14

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 streak­ing 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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Section 2
Kit Specifications

The components provided in the ReadyPrep reduction­alkylation kit are listed below. Each kit contains sufficient
reagent to perform a reduction-alkylation reaction on
100 samples of 300 µl each containing up to 10 mg/ml
of protein.

Items Supplied With Kit

3 ampoules TBP (200 mM), 0.6 ml
3 empty glass vials for storage of TBP after opening the
ampoules
5 amber vials iodoacetamide, 56 mg
1 tube alkylation buffer, 2 ml

Caution!

Care should be exercised when handling the components.

TBP should be used only in areas with good ventilation.
Avoid breathing vapor. Avoid contact with skin. In case of
contact with eyes, flush with water and contact a
physician.

MSDS forms are available upon request.

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Items Required But Not Provided:

• 1.5 ml microcentrifuge tubes

• Microcentrifuge capable of spinning at 12,000–16,000 x g

• Rehydration/sample buffer

Section 3
Storage Conditions

Store the unopened kit at room temperature. Once
opened, follow the storage instructions provided on each
component.

Once an ampoule of TBP has been opened, transfer the
TBP to one of the empty glass vials provided and store
at -20°C.

Iodoacetamide powder should be stored in the dark and is
unstable once in solution. Discard unused iodoacetamide
solution.

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