Chromatrap presentation 2016

© 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
Epigenetic
marks
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 acids
o Phase I and II
• Hydroxamic acids
o 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 antibody
added
3. Reverse cross link to
release DNA from protein
Chip assays:
• Complex
• Difficult
• Slow
• Require skill/experience
Therefore bottleneck
In epigenetic research
Selectively enriched DNA4. PCR or ChIP SEQ to
investigate site specific marks
Proteinase K digestion:

Chromatrap® offers an inert solid-phase
scaffold for better immunoprecipitation
2. Chromatin/antibody complex
binds to protein
Protein A or G
covalently bonded
to matrix
4. Chromatin released into fresh solution3. 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 scaffold
o Inert material
o Open structure
o Surface modified
• Flow through process
Promotes molecular movement
Better sample mixing
Minimises non specific
binding

Available formats
Chromatrap® technology is available for use in several key formats:
• ChIP qPCR with spin columns
• ChIP-seq with spin columns
• HT-ChIP qPCR with 96-well plates
• HT-ChIP-seq with 96-well plates
• Enzymatic ChIP kits for qPCR & seq
• ChIP from FFPE samples
• Native ChIP
• ChIP Validated antibody and primer sets
• DNA purification

A simpler and faster process with less
manual handling
TRADITIONAL METHODS
10 hours total
CHROMATRAP for qPCR
Under 5 h
Typical chip process in the lab
Chromatin
preparation
Shearing
Antibody
binding to
protein
Antibody/
Protein binding
to chromatin
Wash steps to
remove non
specific binding
Elute to
release
chromatin
Reverse
cross link
DNA
purification
PCR or
Chip 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

ChIP-QPCR Benefits
• Reliability and reproducibility at low chromatin loadings
• Best signal-to-noise ratio of any kit at low chromatin loadings
• Protocols based on chromatin concentration, not just cell
numbers
• No DNA clean up required
• High throughput capability

Benchmarking
• Excellent signal strength from 1μg sample size:
• Allows lower input volume
• More assays per sample
• Chromatrap® RECOMMENDS smaller input sample sizes of 50
ng-7000 ng for ChIP qPCR

ChIP-seq
Chromatrap® ChIP Seq kit has been optimized to deliver the greatest
quality and quantity of DNA for sequencing
Chromatrap® routinely obtain
low levels of duplication (<2%)
And high % uniquely mapped
reads (98%)

ChIP Seq Benefits
• Ample DNA to perform library preparation from a single IP
• Positive target enrichment typically 15-20 fold when compared
with background IgG
• ChIP-seq from as little as 1-50µg of chromatin
• Wide dynamic range allows lower chromatin loadings and
minimises antibody requirements
• Reduced IP times
• Fully compatible with Illumina platforms
• Compatible with both qPCR and seq as downstream process
• Offers greater flexibility of chromatin loading for challenging cell
types

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 targets
o 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

Enzymatic ChIP
H3 signal enrichment at the GAPDH promoter..
Enzymatically sheared chromatin
1500 bp
500 bp
100 bp
Chromatrap® Enzymatic Shearing Kit is the perfect cost-effective
alternative to sonication.
• Isolation of good quality chromatin
• Chromatin preparation to ChIP
analysis in one day
• Compatible with qPCR and seq
• Excellent enrichment, independent of
starting cell number

Native ChIP
Chromatrap® Native ChIP Kit is the only commercial method to study
chromatin in it’s ‘native’ state
• No chemical fixation required
• Cells in more natural ‘native’ state
• Uses enzymatic digestion
• Ideal for enrichment of histone
modifications and some abundant
transcription factors
• Compatible with qPCR and seq
• Wide dynamic range suitable for high and
low chromatin loadings
• More IP assays per sample
• Available in spin column and high
throughput
• Excellent enrichment, independent of
starting cell number

FFPE ChIP
Wide dynamic range makes Chromatrap suitable for FFPE samples,
allowing more IPs per sample
Paraffin
removal
Protein – DNA- Complex
extraction
ChIP
by enzymatic digestion
Chromatrap® FFPE kits outperform
when compared to traditional
methods:
• 10x less starting material
• Requires just 10 3 µm sections
• At least 4x better pull down than
competitor kits
• No need to pool samples for
sequencing

FFPE ChIP benefits
Chromatrap® FFPE ChIP kit has overcome the challenges often
associated with FFPE ChIP, by offering a streamlined protocol
providing the users with excellent quality isolated chromatin.
• Works across a range of FFPE samples
• Ideal for tissue samples, biopsies and difficult to lyse samples
• Available in spin column and high throughput format
• Efficient enrichment with high and low abundant targets
• Provides sufficient DNA for high quality library preparations
• Fully compatible with next gen sequencing

DNA purification
• Higher loadings of active material
• Assay times under 5 minutes
• 98 % recovery for a range of samples sizes
• Up to 50µg DNA an be recovered efficiently and quickly
Available in 2 formats ;
Chromatrap® 96 well DNA
purification kit
Chromatrap® 96 Mirco elution
purification kit allows you to clean
and concentrate samples
Chromatrap® provide 96- well high throughput plates for ultra
pure DNA purification

ChIP Validated Antibodies
Chromatrap® are continuing to build their antibody
catalogue, consisting of high quality epigenetic related
antibodies specifically for use in ChIP
The use of high quality and specific ChIP validated
antibodies is essential for the success of a ChIP assay
Chromatrap® ChIP-validated Antibodies can now be
supplied with the correct matching primer sets to cover
all aspects of ChIP validation.
Reduce time spent searching databases and designing
forward and reverse primers
Chromatrap® are also providing an antibody validation
service to validate specific customer antibodies in ChIP

Chromatrap capabilities
Based in the Institute
of Life Sciences
Swansea
Design of bespoke
high throughput
assays
Design of further new
products for molecular
biology
Expert Technical
Support

Chromatrap Contact Info
For prices, orders, payments and delivery:
•Chromatrap Customer Services Team in our
Wrexham, UK office:
•+44 (0) 1978 666222
•e-mail:int.sales@porvair-sciences.com
For technical, applications or scientific issues:
•Chromatrap Technical Support Team at Swansea
University laboratories in the UK:
•Telephone: +44 (0) 7539 743216
•e-mail: support@chromatrap.com

Chromatrap presentation 2016

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