GST pull-down Protocol

GST-PULL DOWN

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冷泉港

protocol

——

GST pull-down Protocol

GST Pull-down

Margret B. Einarson, Elena N. Pugacheva, and Jason R. Orlinick This protocol was adapted from

of

Protein-Protein

Interactions

with

Glutathione-S- Transferase

Fusion

Proteins,

Chapter

6,

in

Protein- Protein

Interactions,

2nd

edition

(eds.

Golemis

and

Adams).

Cold

Spring

Harbor Laboratory Press, Cold Spring Harbor, NY

, USA, 2005.

INTRODUCTION

Glutathione-S- transferase (GST) fusion proteins have had a wide range of applications since their

introduction as tools for synthesis of recombinant proteins in bacteria. One of these applications is

their

use

as

probes

for

the

identification

of

protein-protein

interactions.

The

pull-down

method

described in this protocol is fundamentally similar to immunoprecipitation. Immunoprecipitation

is

based

on

the

ability

of

an

antibody

to

bind

to

its

antigen

in

solution,

and

the

subsequent

purification of the immunocomplex by collection on protein A- or G-coupled beads. Similarly, the

GST

pull-down

is

an

affinity

capture

of

one

or

more

proteins

(either

defined

or

unknown)

in

solution

by

its

interaction

with

the

GST

fusion

probe

protein

and

subsequent

isolation

of

the

complex

by

collection

of

the

interacting

proteins

through

the

binding

of

GST

to

glutathione-coupled beads.

RELATED INFORMATION

An introduction to the use of GST fusion proteins for studying protein-protein interactions can be

found

in

Identification

of

Protein-Protein

Interactions

with

Glutathione-S- Transferase

(GST)

Fusion Proteins.

This protocol is designed to use a 35S-labeled cell lysate as the source for interacting proteins. For

35S-labeling procedures, see Orlinick and Chao (1996) and Spector et al. (1998). Additionally, if

the interacting protein of interest is known to be confined to a specific cellular compartment (e.g.,

the nucleus), a fraction of the cell lysate corresponding to that compartment (e.g., a nuclear extract

[to prepare, see Dignam et al. 1982]) can be used in place of a total cell lysate.

MATERIALS

This procedure may require equipment or reagents for Western analysis, Coomassie blue staining,

and/or silver staining (see Step 13).

Reagents

Cell lysate (unlabeled or labeled with 35S, depending on experimental goal)

This experiment compares GST versus GST fusion protein, so it is necessary to prepare enough

lysate to provide equal amounts of lysate in each reaction. The amount of lysate needed to detect

an interaction is highly variable. Start with lysate equivalent to 1 x 106 to 1 x 107 tissue culture

cells.

GST fusion protein (see Preparation of GST Fusion Proteins)

GST protein

GST pull-down lysis buffer, ice cold

Reagents for SDS-Polyacrylamide Gel Electrophoresis of Proteins (see Step 12)

2X SDS gel-loading buffer

Tris-Cl (50 mM, pH 8.0) containing 20 mM reduced glutathione (optional; for Step 11 only)

GST-PULL DOWN

手册

Equipment

Equipment for SDS-Polyacrylamide Gel Electrophoresis of Proteins (see Step 12)

Gel dryer (optional; see Step 13)

Glutathione- Sepharose beads (store at 4°

C; do not freeze)

Beads are often supplied by commercial vendors in solutions containing alcohols. It is important

to

wash

the

beads

thoroughly

in

GST

pull-down

lysis

buffer

and

to

generate

a

50/50

slurry

of

beads in GST pull-down lysis buffer prior to use.

Microcentrifuge, precooled to 4°

C

Microcentrifuge tubes, 0.5 mL (optional; for Steps only) and 1.5 mL

Needle, small bore, sterile (optional; for Steps only)

Rotator for end-over-end mixing

Water bath, boiling (optional; see Step 10)

X-ray film (optional; see Step 13)

METHOD

1.

Incubate the cell lysate with 50 μL of glutathione

-Sepharose beads (50/50 slurry in lysis buffer)

and 25 μg of GST (NOT the GST fusion probe protein) for 2 h at 4°C with end

-over-end mixing.

Allow

enough

volume

in

the

tube

to

permit

liberal

mixing;

500

μ

l

to 1

mL

is

a

good

starting

point.

This

step

is

designed

to

preclear

from

the

lysate

proteins

that

interact

nonspecifically

with

the

GST moiety or with the beads alone. If the interaction will be detected primarily with antibodies

directed

to

a

candidate

interacting

protein,

it

is

not

absolutely

necessary

to

preclear

the

lysates

with

GST

or

glutathione-Sepharose

beads.

However,

when

35S-labeled

cell

lysates

are

used

to

identify novel protein-protein interactions, these steps can help to reduce background.

When detecting the interacting protein with antibodies to that protein, it is important to include

2.

Centrifuge at 13,000 rpm for 10 sec at 4°

C in a microcentrifuge.

3.

Transfer the supernatant (precleared cell lysate) to a fresh tube.

4.

Set up two tubes containing equal amounts of the precleared cell lysate.

i.

Add 50 μl of glutathione

-Sepharose beads (50/50 slurry in lysis buffer) to each tube.

ii.

Then add GST protein to one tube and the GST fusion probe to the other (~5-

10 μg each).

The

amount

of

protein

added

should

be

equimolar

in

the

two

reactions

(i.e.,

the

final

molar

concentration of GST should be the same as that of the GST probe protein).

5.

Incubate the tubes for 2 h at 4°

C with end-over-end mixing.

6.

Centrifuge the samples at 13,000 rpm for 10 sec at 4°

C in a microcentrifuge.

7.

Transfer the supernatants to fresh microcentrifuge tubes and reserve them for SDS-PAGE (see

Troubleshooting).