TY - JOUR
T1 - Analysis of protein sumoylation.
AU - Hilgarth, Roland S.
AU - Sarge, Kevin D.
PY - 2006/6
Y1 - 2006/6
N2 - The covalent attachment of small ubiquitin-like modifier (SUMO) proteins to specific lysine residues of target proteins, a process termed sumoylation, is a recently discovered protein modification that plays an important role in regulating many diverse cellular processes. For this reason there is significant interest in identifying new sumoylated proteins and the lysine residue(s) within these target proteins where SUMO attachment occurs. Such knowledge will allow determination of the functional consequences of sumoylation through mutation of the relevant sequences. This unit describes two different experimental approaches for ascertaining specific protein sumoylation: the first is based on immunoprecipitation of the protein of interest followed by SUMO immunoblotting. The second involves incubation of the protein (either an in vitro translation product or a purified recombinant protein) in a reconstituted in vitro sumoylation enzymatic reaction followed by visualization of sumoylated protein as a higher than normal molecular-weight band in SDS-PAGE.
AB - The covalent attachment of small ubiquitin-like modifier (SUMO) proteins to specific lysine residues of target proteins, a process termed sumoylation, is a recently discovered protein modification that plays an important role in regulating many diverse cellular processes. For this reason there is significant interest in identifying new sumoylated proteins and the lysine residue(s) within these target proteins where SUMO attachment occurs. Such knowledge will allow determination of the functional consequences of sumoylation through mutation of the relevant sequences. This unit describes two different experimental approaches for ascertaining specific protein sumoylation: the first is based on immunoprecipitation of the protein of interest followed by SUMO immunoblotting. The second involves incubation of the protein (either an in vitro translation product or a purified recombinant protein) in a reconstituted in vitro sumoylation enzymatic reaction followed by visualization of sumoylated protein as a higher than normal molecular-weight band in SDS-PAGE.
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U2 - 10.1002/0471140864.ps1408s44
DO - 10.1002/0471140864.ps1408s44
M3 - Article
C2 - 18429298
AN - SCOPUS:48849089860
SN - 1934-3655
VL - Chapter 14
SP - Unit 14.8
JO - Current protocols in protein science / editorial board, John E. Coligan ... [et al.]
JF - Current protocols in protein science / editorial board, John E. Coligan ... [et al.]
ER -