Purification of DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKcs) from HeLa Cells

Methods Mol Biol. 2022:2444:227-241. doi: 10.1007/978-1-0716-2063-2_14.

Abstract

With a predicted molecular mass of 469 kDa, expression of recombinant DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is challenging. However, DNA-PKcs is relatively abundant in human cells, making it possible to purify the endogenous protein. Here we describe a method to purify DNA-PKcs and its binding partner Ku70/80 from HeLa cells and describe conditions for transfer of DNA-PKcs and other large polypeptides for immunoblotting.

Keywords: DNA-PKcs; HeLa cells; Ku70/80; Protein purification.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Catalytic Domain
  • DNA / chemistry
  • DNA-Activated Protein Kinase* / chemistry
  • DNA-Activated Protein Kinase* / genetics
  • DNA-Activated Protein Kinase* / metabolism
  • DNA-Binding Proteins* / metabolism
  • HeLa Cells
  • Humans
  • Nuclear Proteins / metabolism

Substances

  • DNA-Binding Proteins
  • Nuclear Proteins
  • DNA
  • DNA-Activated Protein Kinase