Bio-Rad ReadyPrep Protein Extraction Kit User Manual

ReadyPrep™Protein Extraction Kit
(Membrane II)

Instruction Manual

Catalog #163-2084

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

Table of Contents

Section 1 Introduction…………………………………...1–2

Section 2 Kit Specifications…………………………......3–5

Section 3 Storage Conditions………………………..........5

Section 4 Reagent Preparation…………………...........6–7

Section 5 Instructions for Use…………………...........8–13

Section 6 Appendix……………..…………….................14

Section 7 References……………..……………...............15

Section 8 Product Information………………............16–17

Section 1
Introduction

The ReadyPrep protein extraction kit (membrane II) is
designed as a simple, rapid and reproducible method to
isolate cellular membranes and then to extract the
associated integral membrane proteins into a solution
compatible with two-dimensional polyacrylamide gel
electrophoresis (2-D PAGE). The ReadyPrep protein extrac­tion kit (membrane II) joins two existing ReadyPrep kits for
isolation of membrane proteins; it is optimized to effectively
isolate more complex proteins that contain more than two
transmembrane domains, or are resistant to extraction by
the ReadyPrep membrane I kit. The signal kit is specific for
isolation of proteins involved in intracellular membrane
trafficking and signaling pathways. All three of these mem­brane kits reduce sample complexity using cellular location
as a way to fractionate. The procedure for the ReadyPrep
protein extraction kit (membrane II) is based on the original
work of Fujiki et al.
fractions from rat liver after carbonate extraction and the
later adaption of the technique by Molloy et al.
Proteomic analysis of E. coli outer membrane proteins.

The kit contains all the reagents necessary for membrane
isolation and membrane-protein extraction and the protocol

1

for the isolation of cellular membrane

2

for

1

can be applied to a wide variety of biological samples, from
animal cells and tissues to yeast, bacteria and plant
tissues. After the cells or tissue are disrupted using an
ultrasonic probe, the extract is incubated in a Membrane
Protein Concentrating Reagent. This high pH buffer strips
away proteins weakly-associated with membranes while
leaving membrane fragments with their associated integral
proteins intact. Ultracentrifugation is used to concentrate
the membranes into a pellet, and the proteins are
solubilized from the washed pellet using an isoelectric
focusing-compatible 2-D rehydration/sample buffer. The
2-D Rehydration/Sample Buffer 1 utilized in the kit is a
strongly chaotropic buffer containing the zwitterionic
detergent ASB-14, making this solution one of the most
powerful solubilizing reagents available for 2-D gel
electrophoresis. The kit also contains the reducing agent
tributylphosphine (TBP), which has been shown to be more
efficient than dithiothreitol in keeping proteins reduced in
2-D PAGE applications.

3

2

Section 2
Kit Specifications

Each ReadyPrep protein extraction kit (membrane II)
provides sufficient reagents to perform 10 extractions each
starting with 100–200 mg of cells or tissue. The procedure
can easily be scaled up to accommodate larger amounts of
cells or tissue. The entire procedure can be completed in
less than 3 hr.

Items Supplied With Kit

• 2-D Rehydration/Sample Buffer 1. 2 vials. Lyophilized.

After reconstituting, each vial contains 10 ml of 7 M urea,
2 M thiourea, 1 % (w/v) ASB-14 detergent, 40 mM Tris
base, and 0.001% Bromophenol Blue.

• Lysis Buffer. 5 pouches. Each pouch reconstitutes to

make 50 ml of Lysis Buffer.

• Membrane Protein Concentrating Reagent. 5 pouch-

es. Each pouch reconstitutes to make 125 ml of mem­brane protein concentrating reagent.

• TBP Reducing Agent. 1 ampoule containing 0.6 ml of

200 mM tributylphosphine (TBP) in 1-methyl­2-pyrrolidinone sealed under nitrogen gas.

• Empty Vial. 1 storage vial for TBP reducing agent.

• Instruction Manual, 1.

3

Items Required But Not Provided

• 2.0 ml microcentrifuge tubes (for example, VWR catalog
#20170-237).

• Disposable round-bottom tubes for plant sample
extraction (for example, VWR catalog # 60818-725).

• ReadyPrep 2-D cleanup kit (Bio-Rad catalog #163-2130)
for plant sample extraction. When working with plant leaf
tissue, removal of interfering compounds, such as
phenolics, is essential for high quality 2-D gels with
minimal horizontal streaking.

• 50 ml conical centrifuge tube(s), disposable, (for
example, VWR catalog #20171-030).

• 2 clean 125–150 ml beakers or bottles plus stirring bars.

• Ultracentrifuge, rotor and tubes capable of spinning at
100,000 x g. Typically, tubes should have ~30 ml
capacity.

• Microcentrifuge capable of spinning at 12–16,000 x g
at 4°C.

• Sonicator

• Benzonase (endonuclease) (for example, Sigma
catalog #E1014 or #E8263)

• 1 M MgCl

2

solution

• Protease inhibitor(s) (optional)

• Carrier ampholytes (for example, Bio-Lyte



3/10

ampholyte, Bio-Rad catalog #163-2094)

4