ChIP grade antibodies: selection and validation. Rachel Imoberdorf, PhD Senior Development Scientist

Transcription

1 ChIP grade antibodies: selection and validation Rachel Imoberdorf, PhD Senior Development Scientist

2 Introduction Antibody selection Antibody validation ChIP at

3 Introduction

4 Chromatin Immunoprecipitation (ChIP) Allows quantitative analysis of Protein-DNA interactions in vivo. 2.50E-03 ChIP: TBP ab818 Localisation of proteins associated with DNA 2.00E E E E-04 ab818 beads 0.00E+00 GAPDH ACT-G MYO-D SERPINA active inactive Follow changes at promoters Map distribution of factor over entire genome

5 Cross-linking Sonication Immunoprecipitation DNA analysis Quantitative PCR Microarray Cloning

6 Data ChIP: H3 tri-methyl K4 (ab8580) Active genes marked by H3 K4 tri-methylation

7 Antibody selection

8 Antibody is the most important player in ChIP Cross-link Sonication Immunoprecipitation DNA analysis

9 Pass rate for ChIP is low Non histone antibodies: 20% (24/120) Histone antibodies: 50% (25/50)

10 Antibody should be fully characterised and specific Affinity purified Specificity tested in: -ELISA -Peptide inhibition western blot Peptide competior: Peptide inhibition western blot H3 tri-methyl K4 (ab8580) none Mono me K4 Di me K4 Di me K9 Tri me K9 Tri me K27 unmodified Tri me K4 H3 trimethyl K4

11 IP is good indicator for success in ChIP Immunoprecipitation TBP antibody (ab28176) Extract Supernatant TBP ab

12 IHC is also good indicator for success in ChIP Protein conformation: Native Denatured Immunohistochemistry TAF1 antibody (ab28450) Technique: ChIP/IP/IHC Western blot

13 Antibodies working in IP or IHC Code Target IP IHC ChIP ab15597 BRM/SMARCA2 ND ab3652 CBP ND ab13939 ab1791 ab9057 ab3752 ab2605 CenPA Histone H3 HP1 a MeCP2 Menin ND ND no Antibodies working in IP or IHC ab5408 RNA polymerase II CTD repeat [4H8] ab12405 Suv39H1 ab818 TBP ND

14 Polyclonal antibodies have higher success rate in ChIP Polyclonal antibodies Monoclonal antibodies + recognise several epitopes - recognise single epitope - batch to batch variation + minimal batch to batch variation

15 Summary Well characterised and specific IP and IHC are good indicators for success in ChIP Polyclonal antibodies have higher success rate in ChIP

16 Antibody validation

17 ChIP using standard conditions Requirement: Control loci (+/-) Relative to Input 1.00E E E E E+00 GAPDH TBP ACT-G PASS MYO-D SERPINA active inactive ab28176 beads Relative to Input 2.50E E E E E E+00 GAPDH ACT-G active TBP OPTIMISE/FAIL MYO-D SERPINA inactive ab12089 beads Relative to Input 5.00E E E E E E+00 GAPDH ACT-G TBP FAIL MYO-D SERPINA active inactive ab24685 beads

18 Antibody concentration should be optimised 1 10ug of antibody for every 25ug of chromatin Antibody: 2ug 10ug Relative to Input 9.00E E E E E E E E E E+00 GAPDH ACT-G TBP MYO-D SERPINA ab12089 beads Relative to Input 9.00E E E E E E E E E E+00 GAPDH ACT-G TBP MYO-D SERPINA ab12089 beads active inactive active inactive

19 Test different washing conditions Salt concentration of last wash: mM NaCl [NaCl]: Relative to Input 3.75E E E E E E mM GAPDH ACT-G TBP MYO-D SERPINA active inactive ab818 beads Relative to Input 3.75E E E E E E mM GAPDH ACT-G active TBP MYO-D SERPINA inactive ab818 beads Washes should be as stringent as possible

20 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 from Abreview

21 Summary Perform standard ChIP experiment Antibody concentration can be optimized Washing conditions can be optimized Test whether target is immunoprecipitated

22 Chromatin immunoprecipitation at Abcam

23 ChIP at Abcam >160 ChIP grade antibodies

24 Abcam ChIP testing program Antibody passes ChIP if: ChIP: SUV39H1 ab Signal specific - Enrichment over background >10x Data and experimental conditions published on datasheet Enrichment relative to Input GAPDH ALDOA MYO-D F8 SAT2 SATa active inactive heterochromatin ab12405 Beads

25 ChIP batch tested antibodies >40 ChIP batch tested antibodies: - Histones - Histone modifications - Other modified proteins Enrichment relative to Input ChIP: Histone H3 (tri methyl K4) ab GAPDH RPL30 ALDOA MYO-D SERPINA AFM ab8580 Beads active inactive Every new batch of this antibody is tested in ChIP.

26 ChIP batch tested Histone modification antibodies Specificity tested by peptide inhibition western blot H3 tri-methyl K4 (ab8580) Peptide competitor: none Mono me K4 Di me K4 Tri me K4 Di me K9 Tri me K9 Tri me K27 unmodified H3 tri-methyl K4 Antibody does NOT cross react

27 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%

28 Primer sequences for ChIP on datasheet

29 ChIP protocol at

30 Technical support for ChIP at Dr Hugh Spotswood

31 Summary Large range of ChIP grade antibodies (>160) In house ChIP testing program Data and experimental conditions published on datasheet ChIP resources

32 The chromatin team Dr Rhian Hayward Marketing manger Candy Smelly Marketing coordinator Dr Jonathan Frampton Business Development Associate Dr Rachel Imoberdorf Senior Development Scientist Dr Hugh Spotswood Technical Support Thank you for your attention!

33 ChIP on ChIP Pokholok et al., (2005) Genome-wide Map of Nucleosome Acetylation and Methylation in Yeast. Cell, 122,

34 In house tested antibody ChIP: Suv39H1(ab12405) SUV39H1 enriched at heterochromatin

35 ChIP batch tested antibodies Stock batch New batch Enrichment relative to Input ChIP: Histone H3 (tri methyl K4) ab GAPDH RPL30 active ALDOA MYO-D SERPINA inactive AFM ab8580 Beads Enrichment relative to Input ChIP: Histone H3 (tri methyl K4) ab GAPDH RPL30 active ALDOA MYO-D FAIL SERPINA AFM inactive ab8580 Beads

36 Sonication efficiency affected by: Foam Cell type Cell density Extent of cross-linking M Sonicated chromatin TIP: Optimise sonication conditions for every cell type and growth condition.