Detection of the DNA binding of transcription factors in situ at the single-cell resolution in cultured cells by proximity ligation assay

Babul Moni Ram; Chengkai Dai

doi:10.1016/j.xpro.2023.102692

Detection of the DNA binding of transcription factors in situ at the single-cell resolution in cultured cells by proximity ligation assay

STAR Protoc. 2023 Dec 15;4(4):102692. doi: 10.1016/j.xpro.2023.102692. Epub 2023 Nov 2.

Authors

Babul Moni Ram¹, Chengkai Dai²

Affiliations

¹ Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702, USA. Electronic address: babul.ram@nih.gov.
² Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702, USA. Electronic address: chengkai.dai@nih.gov.

Abstract

Transcription factors (TFs) play a pivotal role in gene expression, and their DNA binding is the prerequisite to instigating gene transcription. Here, we present a protocol that exploits the proximity ligation assay technique to measure the DNA-binding activities of TFs in situ at the single-cell resolution. We describe steps for immunostaining with specific antibodies against double-stranded DNA and the TFs of interest, probe incubation, proximity ligation, and signal amplification. We then detail procedures for imaging and image analysis. For complete details on the use and execution of this protocol, please refer to Dai et al. (2015)¹ and Xu et al. (2023).².

Keywords: Cancer; Cell Biology; Cell Culture; Gene Expression; Genomics; Microscopy; Molecular Biology; Molecular/Chemical Probes; Single Cell.

Published by Elsevier Inc.

MeSH terms

Antibodies*
Cells, Cultured
Image Processing, Computer-Assisted
Transcription Factors* / genetics

Substances

Transcription Factors
Antibodies

Grants and funding

ZIA BC011767/ImNIH/Intramural NIH HHS/United States