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

Protocol

Macro-Molecular coMplexes

Binary & TerTiary inTeracTions

TransiTion inTo iMaging sTudies

parallel proTein:proTein inTeracTions

More inforMaTion

Protein:Protein Interactionswith the HaloTag® platform

✓ �Identify�more�physiologically- relevant�protein�partners

✓ Much�lower�background

✓ �Transition�from�pull-downs�into�imaging�studies�with�the�same�construct

✓ �Simple�customizable�arrays�for�oriented�protein�immobilization

P r o t e i n : P r o t e i n i n t e r a c t i o n s w i t h t h e h a l o ta g ® P l at f o r m

feaTures & BenefiTs

P roto co l

Macro-Molecular coMplexes

Binary & TerTiary inTeracTions

TransiTion inTo iMaging sTudies

parallel proTein:proTein inTeracTions

More inforMaTion

Mammalian pull-down protocol overview

HaloLink

in vivo formation of protein complexes

2. Passive cell lysis

3. Rapid complex capture 4. Gentle washes

5a. TEV cleavage

or

9696

MA

5b. SDS Elution

6-8 HOURS1-2 Days

HaloTag

HaloTag

HaloTag

HaloTag1. Express HaloTag® fusion bait protein

P r o t e i n : P r o t e i n i n t e r a c t i o n s w i t h t h e h a l o ta g ® P l at f o r m

feaTures & BenefiTs

Protocol

Macro-Molecular co m P l e x es

Binary & TerTiary inTeracTions

TransiTion inTo iMaging sTudies

parallel proTein:proTein inTeracTions

More inforMaTion

Study macro-molecular complexes

Get consistent results and clean controls with Halotag® when pulling down macro-molecular complexes.

the majority (26/29) of known subunits for the transcriptional coactivator complex, Mediator, were specifically isolated in a single pull-down experiment using Med26 as bait.

the Ht control demonstrates the low non-specific binding of this fusion tag in pull-downs since 0/29 subunits of Mediator were identified.

Med26 HT Control

220

120

10080

60

50

40

30

9712

MA

P r o t e i n : P r o t e i n i n t e r a c t i o n s w i t h t h e h a l o ta g ® P l at f o r m

feaTures & BenefiTs

Protocol

Macro-Molecular coMplexes

B i n a ry & t e rt i a ry i n t e r ac t i o n s

TransiTion inTo iMaging sTudies

parallel proTein:proTein inTeracTions

More inforMaTion

expected cytoplasmic binary and tertiary protein interactions in the nfkB pathway were identified with specific p65-HaloTag® pull-down.

Study simple (binary and tertiary) interactions

HaloTag control

P65–HaloTag fusion

9414

TB

Proteins�identified�by�MSrela(p65)relBc-relIkBaIkBbIkBep100p105(p50)p52

P r o t e i n : P r o t e i n i n t e r a c t i o n s w i t h t h e h a l o ta g ® P l at f o r m

feaTures & BenefiTs

Protocol

Macro-Molecular coMplexes

Binary & TerTiary inTeracTions

t r a n s i t i o n i n to i m ag i n g st u d i es

parallel proTein:proTein inTeracTions

More inforMaTion

No�additional�cloning�is�necessary to go between protein pull-down studies and cell imaging applications, as all Halotag® ligands are interchangeable.

Transition from pull-downs into imaging

u2os cells stably expressing p65-HaloTag labeled with HaloTag TMrdirect™ ligand show proper cytoplasmic localization.

9416

TA

A. B.

P r o t e i n : P r o t e i n i n t e r a c t i o n s w i t h t h e h a l o ta g ® P l at f o r m

feaTures & BenefiTs

Protocol

Macro-Molecular coMplexes

Binary & TerTiary inTeracTions

TransiTion inTo iMaging sTudies

Pa r a l l e l P rot e i n:P rot e i n i n t e r ac t i o n s

More inforMaTion

Identify new protein partners faster

a single slide was used to study multiple protein:protein interactions.

JunFKBP

p53Id

hRILuc

HaloTagBlank

ImmobilizedHaloTag®

fusion Proteins

GST-FR

B

GST-Fo

s

JUN:FosFKBP:FRB

9714

MA

Express HaloTag® fusion proteinsin TNT® cell-free lysates ormammalian cell lysates

ImmobilizedHaloTag®

fusion proteins

Spot lysate(no pre-purification needed) Wash

9713

MA

HaloTag

BAIT

HaloTag

BAIT

HaloTag

BAIT

HaloTag

BAIT

P r o t e i n : P r o t e i n i n t e r a c t i o n s w i t h t h e h a l o ta g ® P l at f o r m

feaTures & BenefiTs

Protocol

Macro-Molecular coMplexes

Binary & TerTiary inTeracTions

TransiTion inTo iMaging sTudies

parallel proTein:proTein inTeracTions

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 • 19765 • parT #is032

References gallo, s. et al. (2011) Tagging of functional ribosomes in living cells by HaloTag® technology.In Vitro Cell Dev. Biol. Anim. 47(2):132-8.

neklesa, T.K. et al. (2011) small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. Nat. Chem. Biol. 2011 7(8): 538-43.

Méndez, j. et al. (2010) efficient isolation, identification and labeling of Intracellular Mammalian Protein complexes. [cited: 2011, 01, 28]; 1, tpub_040.

Ordering Information

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

HaloTag®�Mammalian�Pul l -Down�and�label ing�System

24 reactions g6500, g6504

Halolink™�Protein�arrays variousg6140,

g6180, g6190

Halotag and tNT are registered trademarks of promega corporation. Halolink and TMrdirect are trademarks of promega corporation.