A chromatin immunoprecipitation (ChIP) protocol for use in whole human adipose tissue

Yulia Haim, Tanya Tarnovscki, Dana Bashari, Assaf Rudich

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

Chromatin immunoprecipitation (ChIP) has become a central method when studying in vivo protein-DNA interactions, with the major challenge being the hope to capture "authentic" interactions. While ChIP protocols have been optimized for use with specific cell types and tissues including adipose tissue-derived cells, a working ChIP protocol addressing the challenges imposed by fresh whole human adipose tissue has not been described. Utilizing human paired omental and subcutaneous adipose tissue obtained during elective abdominal surgeries, we have carefully identified and optimized individual steps in the ChIP protocol employed directly on fresh tissue fragments. We describe a complete working protocol for using ChIP on whole adipose tissue fragments. Specific steps required adaptation of the ChIP protocol to human whole adipose tissue. In particular, a crosslinking step was performed directly on fresh small tissue fragments. Nuclei were isolated before releasing chromatin, allowing better management of fat content; a sonication protocol to obtain fragmented chromatin was optimized. We also demonstrate the high sensitivity of immunoprecipitated chromatin from adipose tissue to freezing. In conclusion, we describe the development of a ChIP protocol optimized for use in studying whole human adipose tissue, providing solutions for the unique challenges imposed by this tissue. Unraveling protein-DNA interaction in whole human adipose tissue will likely contribute to elucidating molecular pathways contributing to common human diseases such as obesity and type 2 diabetes.

Original languageEnglish
Pages (from-to)E1172-E1177
JournalAmerican Journal of Physiology - Endocrinology and Metabolism
Volume305
Issue number9
DOIs
StatePublished - 1 Nov 2013

Keywords

  • ChIP
  • Endpoint PCR
  • Protein-DNA interaction
  • Transcriptional regulation
  • Whole human adipose tissue

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