InterPlay Mammalian TAP System Instruction Manual

InterPlay Mammalian TAP System

INSTRUCTION MANUAL

Catalog #240103 InterPlay N-terminal Mammalian TAP System

#240104 InterPlay C-terminal Mammalian TAP System

#240101 InterPlay N-terminal Mammalian TAP Vectors

#240102 InterPlay C-terminal Mammalian TAP Vectors

#240107 InterPlay TAP Purification Kit

#240099 InterPlay TAP Purification Buffer Kit

Revision C

For In Vitro Use Only

240101-12

LIMITED PRODUCT WARRANTY

This warranty limits our liability to replacement of this product. No other warranties of any kind,
express or implied, including without limitation, implied warranties of merchantability or fitness for
a particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect,
consequential, or incidental damages arising out of the use, the results of use, or the inability to use
this product.

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InterPlay Mammalian TAP System

CONTENTS

Materials Provided.............................................................................................................................. 1

Storage Conditions.............................................................................................................................. 2

Additional Materials Required .......................................................................................................... 2

Notices to Purchaser ........................................................................................................................... 3

Introduction......................................................................................................................................... 5

InterPlay TAP System Considerations.............................................................................................. 8

Description of the Vectors .................................................................................................................. 9

pNTAP Vector Map ............................................................................................................ 10

pCTAP Vector Map............................................................................................................. 11

Control Vector Maps ........................................................................................................... 12

Preparing the Vector......................................................................................................................... 13

Preparing the pNTAP/pCTAP Expression Vector .............................................................. 13

Ligating the Insert................................................................................................................ 14

Transformation .................................................................................................................... 15

Verification of Insert Size, Orientation, and Percentage ..................................................... 15

Mammalian Cell Transfection ......................................................................................................... 17

Transfecting the Experimental and Control Vectors ........................................................... 17

Detecting Gene Expression ................................................................................................. 18

Preparing the Reagents for the TAP Protocol................................................................................ 19

TAP Protocol ..................................................................................................................................... 21

Preparing the Protein Extracts ............................................................................................. 21

Preparing the Streptavidin Resin ......................................................................................... 22

Purifying the Protein Complexes Using Streptavidin Resin................................................ 22

Preparing the Calmodulin Resin.......................................................................................... 23

Purifying the Protein Complexes Using Calmodulin Resin ................................................ 23

Detection of Purified Proteins .......................................................................................................... 25

Appendix: Concentrating the TAP Protein Complex-containing Supernatant .......................... 26

Troubleshooting ................................................................................................................................ 28

Preparation of Media and Reagents................................................................................................ 29

References .......................................................................................................................................... 30

Endnotes............................................................................................................................................. 30

MSDS Information............................................................................................................................ 30

InterPlay Mammalian TAP System*

ATERIALS PROVIDED

M

Catalog

Materials provided

TAP expression vectors

pNTAP-A expression vector
(1 μg/μl)

pNTAP-B expression vector
(1 μg/μl)

pNTAP-C expression vector
(1 μg/μl)

pCTAP-A expression vector
(1 μg/μl)

pCTAP-B expression vector
(1 μg/μl)

pCTAP-C expression vector
(1 μg/μl)

TAP expression control vectors

pNTAP-Mef2a expression
control vector (1 μg/μl)

pCMV-Tag2-Mef2c expression
control vector (1 μg/μl)

InterPlay TAP purification kit (#240107)a

Lysis buffer 50 ml 50 ml — — 50 ml 50 ml

0.5 M EDTA 200 μl 200 μl — — 200 μl 200 μl

14.4 M β-mercaptoethanol 69 μl 69 μl — — 69 μl 69 μl

Streptavidin resin (#240105)b 1.25 ml 1.25 ml — — 1.25 ml —

Streptavidin binding buffer (SBB) 25 ml 25 ml — — 25 ml 25 ml

Streptavidin elution buffer (SEB) 5 ml 5 ml — — 5 ml 5 ml

Streptavidin supernatant
supplement

MS-Grade calmodulin resin
(#240106)

Calmodulin binding buffer
(CBB)

Calmodulin elution buffer (CEB) 2.5 ml 2.5 ml — — 2.5 ml 2.5 ml

a

The InterPlay TAP purification kit provides reagents sufficient for 5 purifications (1 × 108 cells/purification).

b

The resin volume listed applies to the settled resin only. The resin is supplied as a 50% slurry, bringing the total volume to

twice the amount listed.

b

#240103

20 μg — 20 μg — — —

20 μg — 20 μg — — —

20 μg — 20 μg — — —

— 20 μg — 20 μg — —

— 20 μg — 20 μg — —

— 20 μg — 20 μg — —

30 μg 30 μg 30 μg 30 μg — —

30 μg 30 μg 30 μg 30 μg — —

100 μl 100 μl — — 100 μl 100 μl

0.625 ml 0.625 ml — — 0.625 ml —

40 ml 40 ml — — 42 ml 40 ml

Catalog

#240104

Catalog

#240101

Catalog

#240102

Catalog

#240107

Catalog

#240099

* Patent Pending
Revision C © Agilent Technologies, Inc. 2008.

InterPlay Mammalian TAP System 1

STORAGE CONDITIONS

All vectors: Store at –20°C upon receipt.
All other components: Store at 4°C upon receipt. Do not freeze.

ADDITIONAL MATERIALS REQUIRED

Anti-Calmodulin binding protein epitope tag antibody (Upstate Catalog #07-482)
Anti-FLAG

Mammalian cell transfection reagent
Media for cell growth and transfection
Microcon
Protease inhibitors [e.g., Protease inhibitor cocktail (Sigma Catalog #P8340), PMSF (Sigma

®

M2 antibody (used to detect expression from the pCMV-Tag2-Mef2c control vector;

Stratagene catalog #200471 or 200472)

®

YM-10 centrifugal filter unit (Millipore Catalog #42421)

Catalog #P7626), etc.]

2 InterPlay Mammalian TAP System

NOTICES TO PURCHASER

CMV Promoter

The use of the CMV Promoter is covered under U.S. Patent Nos. 5,168,062 and 5,385,839 owned by
the University of Iowa Research Foundation and licensed FOR RESEARCH USE ONLY.

FLAG® License Agreement

The enclosed DNA expression vector and/or antibody are specifically adapted for a method of
producing selected protein molecules covered by one or more of the following patents owned by
Sigma-Aldrich Co.: U.S. Patent Nos. (5,011,912, 4,703,004, 4,782,137 and 4,851,341;EP Patent No.
150,126 (Austria, Belgium, Switzerland, France, United Kingdom, Italy, Netherlands and Sweden);
EP Patent No. 335,899 (Belgium, Switzerland, Germany, France, United Kingdom, Italy,
Luxembourg and Sweden); German Patent No. P3584260.1; Canadian Patent No. 1,307,752; and
Japanese Patent Nos. 1,983,150 and 2,665,359. Your payment includes a limited license under these
patents to make only the following uses of these products:

A. Vector License: You may use the enclosed vector to transform cells to produce proteins
containing the amino acid sequence DYKDDDDK for research purposes provided, however, such
research purposes do not include binding an unlicensed antibody to any portion of this amino acid
sequence nor using such proteins for the preparation of antibodies having an affinity for any
portion of this amino acid sequence.

B. Antibody License: You may only use the enclosed antibody for research purposes to perform
a method of producing a protein in which the protein is expressed in a host cell and purified by
use of the antibody in accordance with a claim in one of the above patents in force in a country
where the use actually occurs so long as: (1) you perform such method with a DNA expression
vector licensed from Sigma-Aldrich Co.; and (2) you do not bind (or allow others to bind) an
unlicensed antibody to any DYKDDDDK epitope of any fusion protein that is produced by use of
the method.

This license does not include any rights under any other patents. You are not licensed to use the
vector and/or antibody in any manner or for any purposed not recited above. As used above, the term
“unlicensed antibody” means any antibody which Sigma-Aldrich Co. has not expressly licensed
pursuant to Paragraph B, above. Sigma-Aldrich Co. hereby expressly retains all rights in the above
listed patents not expressly licensed hereunder.

If the terms and conditions of this License Agreement are acceptable to you, then you may open the
vessel(s) containing the vector and/or antibody and, through such act of opening a vessel, will have
shown your acceptance to these terms and conditions.

If the terms and conditions of this License Agreement are not acceptable to you, then please return
the vessel(s) unopened to Stratagene for a complete refund of your payment.

For additional licensing information or to receive a copy of any of the above patents, please contact
the Sigma-Aldrich Co. licensing department at telephone number 314-771-5765.

InterPlay Mammalian TAP System 3

SBP Tag

For Research Use Only - Not for any clinical, therapeutic, or diagnostic use in humans or animals.

The purchase of this product conveys to the buyer the limited, non-exclusive, non-transferable right
(without the right to resell, repackage, or sublicense) to use this product solely for research purposes.
No other right or license is granted to the buyer whether expressly, by implication, by estoppel or
otherwise. In particular, the purchase of this product does not include or carry any right or license to
use, develop, or otherwise exploit this product commercially, and no rights are conveyed to the buyer
to use this product or components of this product for any other purposes.

This product is sold pursuant to an agreement with The General Hospital Corporation, and The
General Hospital Corporation reserves all rights relating to this product, except as expressly set forth
above. For information regarding obtaining a license for uses other than research purposes, please
contact The General Hospital Corporation at [phone].

4 InterPlay Mammalian TAP System

INTRODUCTION

Identification of protein–protein interactions is at the core of understanding
biological processes occurring in living cells. Traditionally, potential
interacting proteins have been identified by genetic methods (two–hybrid
screens) with subsequent verification of the interaction by
co-immunoprecipitation. While this method has been successful for
detection of two interacting proteins, it is of limited utility when more
complex protein aggregates such as ribosomes, spliceosome complexes, or
transcription complexes are investigated. To overcome this limitation, an
alternative method was developed for purification of yeast protein
complexes.

1, 2

This tandem affinity purification (TAP) method combines
purification of a protein complex of interest using affinity purification tags
with subsequent mass spectrometry identification of unknown protein
complex components. The key feature of this technology is the use of two
different affinity purification tags that are fused to at least one known
component of the protein complex of interest by genetic methods.
Performing two consecutive purification steps using affinity purification
tags that have gentle washing and elution conditions allows for isolation
without disruption of the targeted complex.

The Stratagene Interplay TAP systems improve upon the original published
protocol with two peptide tags that allow for isolation of exceptionally clean
proteins without disrupting the targeted complex (see Figures 1 and 2). The
SBP tag, a synthetic sequence isolated from a random peptide library, has a
high affinity for the streptavidin resin provided (~2 × 10
effectively eluted with biotin.

3, 4

The CBP tag, derived from a C-terminal

-9

M), and can be

fragment of muscle myocin light-chain kinase, has a high affinity for the

-9

calmodulin resin provided (~1 × 10
removal of calcium with a chelating agent, recovery of the tagged protein
from the resin is achieved.

5–7

Both tags can be eluted from their respective

M) in the presence of calcium. Upon

resins with gentle washing and small molecule elution conditions thus
increasing the amount and purity of the resulting purified protein complex.
Protease digestion is not required to recover the interacting protein partners.

InterPlay Mammalian TAP System 5

We have validated the system by co-transfecting mammalian cells with
vectors containing known interacting proteins. Since members of the
myocin enhancing factor 2 (MEF2) family are known to interact, MEF2a
and MEF2c are used to demonstrate tandem affinity purification.
The pNTAP-Mef2a vector contains MEF2a, tagged at the N-terminus with
SBP and CBP affinity tags. The pCMV-Tag2-Mef2c vector contains MEF2c
with an N-terminal FLAG tag. When co-transfected, the expressed
MEF2 proteins from each vector interact. Following transfection, cells are
harvested and the proteins are purified using streptavidin resin followed by
calmodulin resin (see Figure 2). Gentle washing and elution conditions
allow the protein–protein interactions to remain intact. The purified protein
complex is analyzed by SDS-PAGE and the MEF2c is detected by Western
blotting using an antibody to the FLAG peptide, indicating that it interacted
and co-purified with its partner, MEF2a. The proteins are further
characterized by in-gel digestion with trypsin followed by mass
spectrometry analysis, confirming interaction.

SBP tag MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREPSGGCKLG

CBP tag KRRWKKNFIAVSAANRFKKISSSGAL

FIGURE 1 Amino acid sequences of the streptavidin (SBP) and calmodulin binding peptides (CBP).

6 InterPlay Mammalian TAP System

FIGURE 2 Tandem affinity purification of the tagged protein of interest and interacting proteins using streptavidin resin
followed by calmodulin resin.

InterPlay Mammalian TAP System 7