RT Journal Article
SR Electronic
T1 Silica-based solid-phase extraction of cross-linked nucleic acid–bound proteins
JF Life Science Alliance
JO Life Sci. Alliance
FD Life Science Alliance LLC
SP e201800088
DO 10.26508/lsa.201800088
VO 1
IS 3
A1 Claudio Asencio
A1 Aindrila Chatterjee
A1 Matthias W Hentze
YR 2018
UL https://www.life-science-alliance.org/content/1/3/e201800088.abstract
AB Proteins interact with nucleic acids to regulate cellular functions. The study of these regulatory interactions is often hampered by the limited efficiency of current protocols to isolate the relevant nucleic acid–protein complexes. In this report, we describe a rapid and simple procedure to highly enrich cross-linked nucleic acid–bound proteins, referred to as “2C” for “complex capture.” This method is based on the observation that silica matrix–based columns used for nucleic acid purification also effectively retain UV cross-linked nucleic acid–protein complexes. As a proof of principle, 2C was used to isolate RNA-bound proteins from yeast and mammalian Huh7 cells. The 2C method makes RNA labelling redundant, and specific RNA–protein interactions can be observed and validated by Western blotting. RNA–protein complexes isolated by 2C can subsequently be immunoprecipitated, showing that 2C is in principle compatible with sensitive downstream applications. We suggest that 2C can dramatically simplify the study of nucleic acid–protein interactions and benefit researchers in the fields of DNA and RNA biology.