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Chromatrap presentation 2016

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Chromatrap® is able to reduce the time spent in the laboratory by minimising the need for manual handling. By reducing the time spent using a pipette and with no incubation required, it means that results are achieved faster.

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Chromatrap presentation 2016

  1. 1. © 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
  2. 2. © 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
  3. 3. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  4. 4. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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:
  5. 5. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  6. 6. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  7. 7. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  8. 8. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  9. 9. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  10. 10. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  11. 11. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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%)
  12. 12. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  13. 13. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  14. 14. © Copyright Porvair Filtration Group Limited Commercial In Confidence Chromatrap 96 example: subset of data collected. 4 targets; 4 cell types; 5 target gene loci Shows experimental flexibility
  15. 15. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  16. 16. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  17. 17. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  18. 18. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  19. 19. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  20. 20. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  21. 21. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  22. 22. © Copyright Porvair Filtration Group Limited Commercial In Confidence 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
  23. 23. © 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

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