ChIP grade antibodies: selection and validation

Rachel Imoberdorf, PhDSenior Development Scientist

• Antibody selection

• ChIP at www.abcam.com

• Antibody validation

• Introduction

Introduction

Chromatin Immunoprecipitation (ChIP)

Allows quantitative analysis of Protein-DNA interactions in vivo.

• Localisation of proteins associated with DNA0.00E+00

5.00E-04

1.00E-03

1.50E-03

2.00E-03

2.50E-03

GAPDH

ACT-G

MYO-D

SERPINA

ab818beads

ChIP: TBP ab818

active inactive

• Follow changes at promoters

• Map distribution of factor over entire genome

Cross-linking DNA analysis

Quantitative PCR

Microarray

Cloning

Sonication Immunoprecipitation

www.abcam.com/chip

ChIP: H3 tri-methyl K4 (ab8580)

● Active genes marked by H3 K4 tri-methylation

Data

Antibody selection

Antibody is the most important player in ChIP

• Cross-link

• Sonication

• Immunoprecipitation

• DNA analysis

• Non histone antibodies: 20% (24/120)

• Histone antibodies: 50% (25/50)

Pass rate for ChIP is low

Antibody should be fully characterised and specific

none

Mon

o m

e K4

Di m

e K4

Tri m

e K4

Di m

e K9

Tri m

e K9

Tri m

e K2

7un

mod

ified

Peptide competior:

H3 tri-methyl K4

H3 tri-methyl K4 (ab8580)Peptide inhibition western blot• Affinity purified

• Specificity tested in:

-ELISA

-Peptide inhibition western blot

IP is good indicator for success in ChIP

Extra

ctSu

pern

atan

tTB

P ab

Immunoprecipitation –TBP antibody (ab28176)

IHC is also good indicator for success in ChIP

Denatured

Western blot ChIP/IP/IHC

Native Proteinconformation:

Technique:

Immunohistochemistry –TAF1 antibody (ab28450)

Antibodiesworking in IP or IHC

yesNDyesTBPab818

yesyesyesSuv39H1ab12405

yesyesyesRNA polymerase II CTD repeat [4H8]ab5408

yesyesyesMeninab2605

yesyes NDMeCP2ab3752

noyesNDHP1 aab9057

yesyesyesHistone H3ab1791

yesyesyesCenPAab13939

yesyesNDCBPab3652

yesNDyesBRM/SMARCA2ab15597

ChIPIHCIPTargetCode

Antibodies working in IP or IHC

Polyclonal antibodies Monoclonal antibodies

- batch to batch variation + minimal batch to batch variation

Polyclonal antibodies have higher success rate in ChIP

+ recognise several epitopes - recognise single epitope

Summary

• Well characterised and specific

• IP and IHC are good indicators for success in ChIP

• Polyclonal antibodies have higher success rate in ChIP

Antibody validation

ChIP using standard conditions

• Requirement: Control loci (+/-)

0.00E+00

2.50E-03

5.00E-03

7.50E-03

1.00E-02

GAPDH

ACT-G

MYO-D

SERPIN

A

ab28176beads

Rel

ativ

e to

Inpu

t

active inactive

TBP

0.00E+00

5.00E-04

1.00E-03

1.50E-03

2.00E-03

2.50E-03

GAPDH

ACT-G

MYO-D

SERPINA

ab12089beads

Rel

ativ

e to

Inpu

t

active inactive

TBP

0.00E+00

1.00E-04

2.00E-04

3.00E-04

4.00E-04

5.00E-04

GAPDH

ACT-G

MYO-D

SERPIN

A

ab24685beads

Rel

ativ

e to

Inpu

t

active inactive

TBP

PASS

FAIL

OPTIMIS

E/FAIL

• 1–10ug of antibody for every 25ug of chromatin

Antibody concentration should be optimised

2ugAntibody:

0.00E+001.00E-022.00E-023.00E-024.00E-025.00E-026.00E-027.00E-028.00E-029.00E-02

GAPDH

ACT-G

MYO-D

SERPIN

A

ab12089beads

Rel

ativ

e to

Inpu

t

active inactive

TBP

10ug

0.00E+001.00E-022.00E-023.00E-024.00E-025.00E-026.00E-027.00E-028.00E-029.00E-02

GAPDH

ACT-G

MYO-D

SERPIN

A

ab12089beads

Rel

ativ

e to

Inpu

t

active inactive

TBP

Test different washing conditions

• Salt concentration of last wash: 150-500mM NaCl

0.00E+00

7.50E-04

1.50E-03

2.25E-03

3.00E-03

3.75E-03

GAPDH

ACT-G

MYO-D

SERPINA

ab818beads

Rel

ativ

e to

Inpu

t

active inactive

[NaCl]: 500mMTBP

0.00E+00

7.50E-04

1.50E-03

2.25E-03

3.00E-03

3.75E-03

GAPDH

ACT-G

MYO-D

SERPIN

A

ab818beads

Rel

ativ

e to

Inpu

t

active inactive

250mMTBP

• Washes should be as stringent as possible

Western blot can be used to test whether the target has been immunoprecipitated

• Perform ChIP up to the final wash

• Boil samples in SDS loading buffer and resolve on SDS page gel

*Picture fromAbreview

*

Summary

• Perform standard ChIP experiment

• Antibody concentration can be optimized

• Washing conditions can be optimized

• Test whether target is immunoprecipitated

Chromatin immunoprecipitationat Abcam

• >160 ChIP grade antibodies

ChIP at Abcam

• Antibody passes ChIP if:

- Signal specific

- Enrichment over back-ground >10x

0

0.01

0.02

0.03

0.04

GAPDH

ALDOA

MYO-D F8

SAT2

SATa

ab12405Beads

active inactive hetero-chromatin

ChIP: SUV39H1 ab12405

Enric

hmen

t rel

ativ

e to

Inpu

t

• Data and experimental conditions published on datasheet

Abcam ChIP testing program

• >40 ChIP batch tested antibodies:

- Histones

- Histone modifications

- Other modified proteins

0

0.05

0.1

0.15

0.2

0.25

0.3

GAPDHRPL30ALDOAMYO-D

SERPINA

AFM

ab8580Beads

active inactive

ChIP: Histone H3 (tri methyl K4) ab8580

Enric

hmen

t rel

ativ

e to

Inpu

t

Every new batch of this antibody is tested in ChIP.

ChIP batch tested antibodies

none

Mon

o m

e K4

Di m

e K4

Tri m

e K4

Di m

e K9

Tri m

e K9

Tri m

e K2

7un

mod

ified

Peptide competitor:

H3 tri-methyl K4

H3 tri-methyl K4 (ab8580)

• Specificity tested by peptide inhibition western blot

• Antibody does NOT cross react

ChIP batch tested Histone modification antibodies

ChIP: H3 tri-methyl K4 (ab8580) • Antibody passes ChIP if:

- Signal strength comparable to original batch

- Pattern of enrichment over a set of 6 loci similar to original batch

• ChIP batch testing pass rate: 75%

ChIP batch tested Histone modification antibodies

Primer sequences for ChIP on datasheet

ChIP protocol at www.abcam.com/ChIP

Dr Hugh Spotswood

Technical support for ChIP at www.abcam.com

Summary

• In house ChIP testing program

• Large range of ChIP grade antibodies (>160)

• Data and experimental conditions published on datasheet

• ChIP resources

Dr Rachel ImoberdorfSenior Development Scientist

Dr Hugh SpotswoodTechnical Support

Dr Jonathan FramptonBusiness Development Associate

The chromatin team

Candy SmellyMarketing coordinator

Dr Rhian HaywardMarketing manger

Thank you for your attention!

● Pokholok et al., (2005) Genome-wide Map of Nucleosome Acetylation and Methylation in Yeast. Cell, 122, 517–527

ChIP on ChIP

In house tested antibody

ChIP: Suv39H1(ab12405)

● SUV39H1 enriched at heterochromatin

0

0.05

0.1

0.15

0.2

0.25

0.3

GAPDHRPL30ALDOAMYO-D

SERPINA

AFM

ab8580Beads

active inactive

ChIP: Histone H3 (tri methyl K4) ab8580

Enric

hmen

t rel

ativ

e to

Inpu

t

Stock batch New batch

FAIL

ChIP batch tested antibodies

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0.0007

0.0008

GAPDHRPL30ALDOAMYO-D

SERPINA

AFM

ab8580Beads

ChIP: Histone H3 (tri methyl K4) ab8580

Enric

hmen

t rel

ativ

e to

Inpu

t

active inactive

Sonication efficiency affected by:

• Extent of cross-linking

• Cell density

• Cell type

TIP: Optimise sonication conditions for every cell type and growth condition.

• FoamM

2036

1018

500

344

220154

Sonicated chromatin