Download - © Copyright Porvair Filtration Group Limited Commercial In Confidence © Copyright Porvair Filtration Group Limited Commercial In Confidence A more efficient,

© Copyright Porvair Filtration Group Limited Commercial In Confidence© Copyright Porvair Filtration Group Limited Commercial In Confidence

A more efficient, sensitive & robust method of chromatin immunoprecipitation

© Copyright Porvair Filtration Group Limited Commercial In Confidence

Epigenetics: the study of the organisation and management of the genome

Nucleosomes

Histone Tails

Epigenetic landscape

ME

CHROMOSOME

Proteins around which DNA is wound

Epigenetic marks are left by external factors such as:

• Environment• Ageing• Diet• Drugs

There are seven types of epigenetic mark in all including:

• DNA methylation• Histone modifications• Nucleosome positioning• MicroRNA

Epigenetic marks

• Are reversible• Will affect the availability of

genes to be activated• Will affect gene expression

Will affect health

outcomes


Example: Epigenetics in cancer research

Epigeneticmarks

CHROMOSOME

Normal

Abnormal

Abnormal methylation of histone tails typically seen in

cancer

Me

MeMe

Me

MeMe

Normal

Abnormal

Changes in histone acetylation typically seen in cancer

Disease development research examples:

HDAC inhibitors• Cyclical tetrapeptides

o Preclinical to phase II

• Short chain fatty acidso Phase I and II

• Hydroxamic acidso Preclinical to Phase II

Me

ME


Effective chromatin immunoprecipitation (“ChIP”) is critical to epigenetic research

CHROMOSOME

Chip isolates specific epigenetic sites:

1. Chromatin sheared into fragments

2. Specific antibodyadded

3. Reverse cross link to release DNA from protein

Chip assays:

• Complex• Difficult• Slow• Require skill/experience

Therefore bottleneckIn epigenetic research

Selectively enriched DNA4. PCR or ChIP SEQ to investigate site specific marks

Proteinase K digestion:


Targeting the Epigenetic landscape

Complex, reversible context to gene expression and disease process regulation

DNA methylation

5-hydroxy-methylctosine

Histone variants

Architecture proteins CTCF/Cohesin

Non coding RNA (long non coding RNA)

Remodelling complexesHistonemodifications

CTCF

DNA

Seven epigenetic marks:

ncRNA

Remodeller

Me Me

P Me

AcMe

HDACi DNMTi HATi HMTi

Enhanced understanding of aberrant disease mechanisms leads to targeted therapies (DNMT inhibitors, HDAC Inhibitors (Phase 1 trials)

Multiple targets, from readers, writers and erasers to manipulate the chromatin landscape and reprogram disease gene expression profiles


Chromatrap offers an inert solid-phase scaffold for better immunoprecipitation

2. Chromatin binds to proteinProtein A or G

covalently bonded to matrix

4. Chromatin released into fresh solution

3. Everything else is flushed away

1. Chromatin/antibody complex flows through matrix


High surface area and flow-through characteristics are key to efficiency

• Solid phase scaffoldo Inert materialo Open structureo Surface modified

• Flow through process

Promotes molecular movement Better sample mixing

Minimises non specific binding


A simpler and faster process with less manual handling

TRADITIONAL METHODS

10 hours total

CHROMATRAP

5 hours total

Typical chip process in the lab

Chromatin preparation

ShearingAntibody binding to

protein

Antibody/Protein binding

to chromatin

Wash steps to remove non

specific binding

Elute to release

chromatin

Reversecross link

DNA purification

PCR orChip Seq

1 hour

2 - 4 hours

3 simple wash-

through steps. No incubation

5 - 15 wash steps

requiring manual pipette

handling and

incubation

Simple centrifugation

Requires re-suspension and bead

handling by manual pipette

handling

Not required

Separate columns, further

manual handling. Loss of

DNA inevitable


More efficient, sensitive and robust assays

More efficient in the lab

• A faster, simpler process • Less manual handling • Chromatrap 96 allows automation

Better assay performance

• Robust signal to noise• Wide dynamic range of sample sizeo Designed for 1ug sample sizes

• High Sensitivityo Suitable for low abundant targetso Suitable for more challenging cell types


Robust signal to noise compared to traditional methods

Robust signal: noise ratios Excellent DNA enrichment Good replication

Chemukhin et al., 2011; BioVyon Protein A, an alternative solid-phase affinity matrix for chromatin immunoprecipitation. Analytical Biochemistry . 15;412(2):183-8


Wide dynamic range: suitable for all sample sizes

Binding shown for high abundant targets• RNA pol II onto GAPDH

Optimum range shown here: 100ng to 2000ng

Full Chromatrap range: 50ng to 7500ng

Signal to noise clear across dynamic range


Ideal for low chromatin loadings: offers significant assay flexibility advantages

Excellent signal strength from 1µg sample size:

Allows lower volume inputsMore assays per sampleGood results from smaller biopsies

1µg sample:

Chromatrap RECOMMENDS smaller input sample sizes of 0.5µg- 7.0µg


Signal strength: clear differentiation across epigenetic landscape

% Real Signal (Input) in K562


Signal strength allows clear results from difficult or low abundant targets

Excellent correlation between H3K4me3 and H3k27me3 over positive and negative gene targets

% Real Signal (Input) HepG2


High sensitivity allows clear results from challenging cell types

Peripheral blood mononuclear cells: CD14+ and lymphocytes

Diluted Blood

Ficoll®

Platelet + Plasma

PBMC

Ficoll® + Gran.

RBC

Pre-Spin Post-Spin

Difficult to isolate samples from blood: Good signal:noise H3 onto GAPDH


High sensitivity allows clear results for multiple epigenetic targets and multiple gene loci using small samples

Endometrial Differentiation

Pipelle Biopsy1mm sample

Mixed cell population. Stromal and epithelial compartments

Digestion: collagenase DNAase


Chromatrap 96: high throughput with speed and sensitivity

Example plate layout for 96 reactions:

• Up to 96 reactions processed simultaneously, all in one day

• Compatible with automated liquid handling

Allows, for the first time:

• Multiple antibody and gene targetso Parallel investigation of widespread

effects

• Large, reliable data sets collected efficiently

• Multiple cell types tested simultaneously

• Multiple samples tested simultaneously


Chromatrap 96 example: subset of data collected.4 targets; 4 cell types; 5 target gene loci

Shows experimental flexibility


Wide dynamic range and sensitivity allows ChIP-seq

At least 10 ng of DNA is needed for Illumina compatible library preparation. Chromatrap exceeds this even from samples of less than 1000ng:

500ng 1000ng 2000ng 7000ng02468

10121416

% Real Signal (Input)

DNA pull-down range: 50ng 700ng


Wide dynamic range makes Chromatrap suitable for FFPE samples

Prepare high-quality chromatin

Paraffinremoval

Protein – DNA- Complexextraction

ChIP

by enzymatic digestion

Rat GAPDH0

5

10

15

20

25

30

% Real Signal (Input)

Chromatrap

Competitor

FFPE: results

FFPE rat uterine tissue


Chromatrap team capabilities

Based in the Institute of Life Sciences Swansea

Design of bespoke high throughput assays

Design of protocols for challenging cell types

Antibody validation

Protocol optimisation