Expression and evaluation of RING finger proteins.
RING finger proteins represent the largest class of potential ubiquitin ligases. This chapter describes methods used to express and assess the activity of proteins containing RING fingers based on our experience with a number of different family members. In addition to general protocols for assessing activity, specific protocols are provided for evaluating the ubiquitylation of p53 by the RING finger E3 Hdm2/Mdm2. Use of these methods may help identify new E3s, dissect factors involved in ubiquitylation of substrates, and screen for molecules that affect ubiquitylation.
Yang
Yili
Y
Dynamics and Signaling, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
Lorick
Kevin L
KL
Jensen
Jane P
JP
Weissman
Allan M
AM
D000818:Animals; D045744:Cell Line, Tumor; D005978:Glutathione; D006801:Humans; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics
D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D005978:Glutathione