A bifunctional antibody conjugate marks the location of DNA binding proteins on deproteinized DNA fibers

Author:

Pokharel Durga,Shaik Althaf,Gali Himabindu,Ling Chen,Bellani Marina A.,Seidman Michael M

Abstract

ABSTRACTImmunofluorescent foci of DNA Damage Response (DDR) proteins serve as surrogates for DNA damage and are frequently interpreted as denoting specific lesions. For example, Double Strand Breaks (DSBs) are potent inducers of the DDR, whose best-known factor is the phosphorylated histone variant H2AX (γ-H2AX). The association with DSBs is so well established that the reverse interpretation that γ-H2AX invariably implies DSBs is routine. However, this conclusion is inferential and has been challenged. The resolution of this question has been hampered by the lack of methods for distinguishing the location of DDR proteins relative to DSBs caused by sequence indifferent agents. Here, we describe an approach for marking the location of DDR factors in relation to DSBs on DNA fibers. We synthesized a two-arm “Y” conjugate containing biotin and trimethylpsoralen (TMP) coupled to a secondary antibody. After exposure to a DNA breaker, permeabilized mammalian cells were incubated with a primary antibody against the DDR factor followed by binding of the secondary antibody in the conjugate to the primary antibody. Exposure to longwave UV light covalently linked the psoralen to the DNA. DNA fibers were spread, and the immunofluorescence of the biotin tag denoted the location of the target protein.Abstract FigureGraphical abstract

Publisher

Cold Spring Harbor Laboratory

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3