Spike-In Normalization of ChIP Data Using DNA–DIG–Antibody Complex

Eberle, Andrea Brigitte (2018). Spike-In Normalization of ChIP Data Using DNA–DIG–Antibody Complex. In: Visa, Neus; Jordán-Pla, Antonio (eds.) Chromatin Immunoprecipitation: Methods and Protocols. Methods in Molecular Biology: Vol. 1689 (pp. 227-238). New York, NY: Springer New York 10.1007/978-1-4939-7380-4_18

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Chromatin immunoprecipitation (ChIP) is a widely used method to determine the occupancy of specific proteins within the genome, helping to unravel the function and activity of specific genomic regions. In ChIP experiments, normalization of the obtained data by a suitable internal reference is crucial. However, particularly when comparing differently treated samples, such a reference is difficult to identify. Here, a simple method to improve the accuracy and reliability of ChIP experiments by the help of an external reference is described. An artificial molecule, composed of a well-defined digoxigenin (DIG) labeled DNA fragment in complex with an anti-DIG antibody, is synthesized and added to each chromatin sample before immunoprecipitation. During the ChIP procedure, the DNA–DIG–antibody complex undergoes the same treatments as the chromatin and is therefore purified and quantified together with the chromatin of interest. This external reference compensates for variability during the ChIP routine and improves the similarity between replicates, thereby emphasizing the biological differences between samples.

Item Type:

Book Section (Book Chapter)

Division/Institute:

08 Faculty of Science > Departement of Chemistry and Biochemistry

UniBE Contributor:

Eberle, Andrea Brigitte

Subjects:

500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry

ISBN:

978-1-4939-7379-8

Series:

Methods in Molecular Biology

Publisher:

Springer New York

Language:

English

Submitter:

Christina Schüpbach

Date Deposited:

23 Nov 2017 10:37

Last Modified:

23 Nov 2017 10:37

Publisher DOI:

10.1007/978-1-4939-7380-4_18

BORIS DOI:

10.7892/boris.106731

URI:

https://boris.unibe.ch/id/eprint/106731

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