Purification of large cytosolic proteases for in vitro assays: 20S and 26S proteasomes
| Autor: | Stefan, Tenzer, Tobias, Hain, Hendrik, Berger, Hansjörg, Schild |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Glycerol
Proteasome Endopeptidase Complex Erythrocytes Blotting Western Chemical Fractionation Chromatography Ion Exchange Acetylcysteine Cytosol Ammonium Sulfate Proteolysis Chromatography Gel Chemical Precipitation Humans Electrophoresis Polyacrylamide Gel Adsorption Dialysis Oligopeptides Proteasome Inhibitors Ultracentrifugation Enzyme Assays |
| Zdroj: | Methods in molecular biology (Clifton, N.J.). 960 |
| ISSN: | 1940-6029 |
| Popis: | Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and efficiency of antigen processing. Here, we describe the purification of active human 20S and 26S proteasomes from human erythrocytes by DEAE-ion exchange chromatography, ammonium sulfate precipitation, glycerol density gradient centrifugation, and Superose-6 size exclusion chromatography and their characterization using fluorogenic substrates and specific inhibitors. |
| Databáze: | OpenAIRE |
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