rachel imoberdorf, phd senior development scientist · chromatin immunoprecipitation (chip) allows...
TRANSCRIPT
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