Accedi

UT Health Science Center San Antonio

2 ARTICLES PUBLISHED IN JoVE

Biology

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

¹Center for Childhood Cancer and Blood Diseases, Nationwide Children's Hospital

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

JoVE Core

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Yohei Niikura^*1, Lei Fang^*2, Risa Kitagawa^*3, Peizhao Li¹, Yao Xi¹, Ju You¹, Yan Guo², Katsumi Kitagawa³

¹MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, ²Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, ³Greehey Children's Cancer Institute, Department of Molecular Medicine, UT Health Science Center San Antonio

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.