F eat u r es & B e n eF i ts

Background

ligand chemistry

covalent Binding

single fusion construct

more information

HaloTag® TechnologyDriving mammalian protein expression forward

✓ Covalent binding of interchangeable synthetic ligands to fusion tag

✓ Use a single clone for protein purification, pull-down, and imaging studies

✓ Higher recovery, purity, functionality and solubility compared to other fusion tags

Compared to other tags:

• higher capacity than flag®

• cleaner than his6

• no leaching (unlike gst)

• more flexible than gfP

H a l o ta g ® t e c H n o l o g y

features & Benefits

Bac kg ro u n d

ligand chemistry

covalent Binding

single fusion construct

more information

N

T r p 1 0 7

H N A s n 4 1 H

C l

R C A s p 1 0 6

O - O

H N

N H O

C O

G l u 1 3 0

H i s 2 7 2

O H

1 3

2 l

N

T r p 1 0 7

H N A s n 4 1 H

C l

C O O

O H

-O C

O Glu1 3 0

A s p 1 0 6

R

H

P h e 2 7 2

Catalytic process Facilitated bond formation

9755

MA

Origin and optimizationOptimized for mammalian protein expression

dhaaorigin: Rhodococcus rhodochrous Function: forms a covalent intermediate at his272 with chloroalkane substrates by removing cl- upon hydrolysis with water.

Halotag®

optimization: his272Phe mutation allows formation of a stable covalent bond. additional aa changes were made in dhaa to further optimize fusion tag performance.

result: a fusion tag which rapidly forms a covalent attachment to synthetic chloroalkane-based ligands. ideal for protein purification, pull-downs and imaging.

H a l o ta g ® t e c H n o l o g y

features & Benefits

Background

l i g a n d cH e m i st ry

covalent Binding

single fusion construct

more information

HaloTag® ligand chemistry allows interchangeable functionality

• the reactive linker is identical between all halotag® ligands

• move fluidly between protein purification, pull-downs and imaging assays by switching to a new functional group.

Functional group

HaloTag

Proteinof interest

+

Cl

Functional group

HaloTag

Proteinof interest

9756

MA

H a l o ta g ® t e c H n o l o g y

features & Benefits

Background

ligand chemistry

cova l e n t B i n d i n g

single fusion construct

more information

HaloTag® structure and the site of the covalent bond

4787TE

HaloTag® TMRLigand

N-terminus

C-terminus

TMR(functional group)

Chloroalkane(reactive linker)

Phe272

Site of covalent interaction

H a l o ta g ® t e c H n o l o g y

features & Benefits

Background

ligand chemistry

covalent Binding

s i n g l e F u s i o n co n st r u c t

more information

HaloTag® ligandsMany functionalities from a single fusion construct

R

HaloTag®

LigandHaloTag®

Fluorescent ligands

Surfaces

Reactive ligands

9699

MA

oore

Surfac

FFllu

S

solid surfaces• nonmagnetic resin • magnetic beads• customizable arrays

Fluorescent ligands• multiple fluorophores• cell-permeable ligands• cell-impermeable ligands

reactive & capture ligands• introduce novel functionalities

with succinimidyl ester, amine, iodoacetamide and thiol reactive groups

• tag and capture with biotin affinity groups

H a l o ta g ® t e c H n o l o g y

features & Benefits

Background

ligand chemistry

covalent Binding

single fusion construct

m o r e i n Fo r m at i o n

Promega corPoration • 2800 Woods holloW road • madison, Wi 53711-5399 usa • telePhone 608-274-4330 www.promega.com • ©2011 Promega corPoration • all rights reserved • Prices and sPecifications suBject to change Without Prior notice • Printed in usa 2/11 • 19764 • Part #is031

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P r o d u c t s i z e c ata l o g #

Halotag® cloning starter system 1 each g6050

val idated Halotag® human orFs 20µg dna kazusa fhc clones

halotag is a registered trademark of Promega corporation. halolink is a trademark of Promega corporation.

flag is a registered trademark of sigma aldrich.

Protein purification…

Protein interactions…

cellular imaging and labeling…