Purification of the mitotic checkpoint complex (MCC) and the anaphase promoting complex/cyclosome (APC/C) from HeLa cells.
In this protocol, active anaphase promoting complex/cyclosome (APC/C) and its inhibitor, mitotic checkpoint complex (MCC), are purified from HeLa cell extracts. The two complexes are first separated by gel filtration and ion exchange chromatography. The MCC is further purified by an additional gel filtration step. The APC/C activity and the ability of MCC to inhibit the APC/C are assayed at every stage of the purification procedure by in vitro ubiquitination assays. The composition and distribution of the complexes are monitored by Western blot analysis.
Valery
V
Sudakin
D064173:Anaphase-Promoting Complex-Cyclosome; D001671:Biochemistry / Q000379:methods*; D018797:Cell Cycle Proteins / Q000302:isolation & purification*; D002457:Cell Extracts; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D043743:Ubiquitin-Protein Ligase Complexes / Q000302:isolation & purification*
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome