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POSTER PRESENTATION Open Access Systematic optimization of parameters involved in preparation of chromatin and chromatin immunoprecipitation (ChIP) workflow John M Rosenfeld * , Tracy Cooke, Zirong Li, Kan Saito, Konstantin Taganov, Bhaskar Thyagarajan, Alejandra Solache From Epigenetics & Chromatin: Interactions and processes Boston, MA, USA. 11-13 March 2013 The ChIP protocol is a fundamental tool for discoveries in the area of epigenomics and gaining insight into epigenetic phenomenon. Despite the fact that many vendors of life science research tools offer products to support ChIP, very little innovation has occurred around the ChIP method itself to make this process more reproducible and reliable. Issues surrounding antibody specificity and performance are critical to high resolution genomic mapping of chro- matin events. In addition, reliable high-throughput options are not readily available for ChIP despite the ability to automate parts of the workflow. Chromatin preparation, for example, remains a laborious process and the field lacks accepted criteria to make determinations on the quality of chromatin suitable for ChIP. We have engaged in an iterative process of examining parameters that influence chromatin fragmentation on a variety of devices and under various buffer systems, and have developed a protocol for optimizing this process for researchers new to the area of chromatin biology. As a leading antibody supplier, we have produced tools that allow researchers to evaluate the specificity of histone modifications antibodies in their own laboratories utilizing a simple dot blot western blotting approach. In addition, we have developed a new high-throughput and low-cell capable protocol that simplifies the process, reduces varia- bility and significantly improves signal-to-noise ratio. This protocol is particularly suited for new users/beginners, and can utilize chromatin derived from cells and tissue. We present data comparing the effect of different sonication protocols on ChIP, analysis of user-to-user variation, as well as methods to optimize and control for factors that introduce variability in ChIP. Published: 8 April 2013 doi:10.1186/1756-8935-6-S1-P122 Cite this article as: Rosenfeld et al.: Systematic optimization of parameters involved in preparation of chromatin and chromatin immunoprecipitation (ChIP) workflow. Epigentics & Chromatin 2013 6 (Suppl 1):P122. Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color figure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Epigenetics R&D, EMD Millipore, Temecula, CA, USA Rosenfeld et al. Epigentics & Chromatin 2013, 6(Suppl 1):P122 http://www.epigeneticsandchromatin.com/content/6/S1/P122 © 2013 Rosenfeld et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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POSTER PRESENTATION Open Access

Systematic optimization of parameters involvedin preparation of chromatin and chromatinimmunoprecipitation (ChIP) workflowJohn M Rosenfeld*, Tracy Cooke, Zirong Li, Kan Saito, Konstantin Taganov, Bhaskar Thyagarajan, Alejandra Solache

From Epigenetics & Chromatin: Interactions and processesBoston, MA, USA. 11-13 March 2013

The ChIP protocol is a fundamental tool for discoveries inthe area of epigenomics and gaining insight into epigeneticphenomenon. Despite the fact that many vendors of lifescience research tools offer products to support ChIP, verylittle innovation has occurred around the ChIP methoditself to make this process more reproducible and reliable.Issues surrounding antibody specificity and performanceare critical to high resolution genomic mapping of chro-matin events. In addition, reliable high-throughput optionsare not readily available for ChIP despite the ability toautomate parts of the workflow. Chromatin preparation,for example, remains a laborious process and the fieldlacks accepted criteria to make determinations on thequality of chromatin suitable for ChIP.We have engaged in an iterative process of examining

parameters that influence chromatin fragmentation on avariety of devices and under various buffer systems, andhave developed a protocol for optimizing this process forresearchers new to the area of chromatin biology. As aleading antibody supplier, we have produced tools thatallow researchers to evaluate the specificity of histonemodifications antibodies in their own laboratories utilizinga simple dot blot western blotting approach. In addition,we have developed a new high-throughput and low-cellcapable protocol that simplifies the process, reduces varia-bility and significantly improves signal-to-noise ratio. Thisprotocol is particularly suited for new users/beginners, andcan utilize chromatin derived from cells and tissue. Wepresent data comparing the effect of different sonicationprotocols on ChIP, analysis of user-to-user variation, aswell as methods to optimize and control for factors thatintroduce variability in ChIP.

Published: 8 April 2013

doi:10.1186/1756-8935-6-S1-P122Cite this article as: Rosenfeld et al.: Systematic optimization ofparameters involved in preparation of chromatin and chromatinimmunoprecipitation (ChIP) workflow. Epigentics & Chromatin 2013 6(Suppl 1):P122.

Submit your next manuscript to BioMed Centraland take full advantage of:

• Convenient online submission

• Thorough peer review

• No space constraints or color figure charges

• Immediate publication on acceptance

• Inclusion in PubMed, CAS, Scopus and Google Scholar

• Research which is freely available for redistribution

Submit your manuscript at www.biomedcentral.com/submit

Epigenetics R&D, EMD Millipore, Temecula, CA, USA

Rosenfeld et al. Epigentics & Chromatin 2013, 6(Suppl 1):P122http://www.epigeneticsandchromatin.com/content/6/S1/P122

© 2013 Rosenfeld et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.