6533b7ddfe1ef96bd127475b

RESEARCH PRODUCT

Purification of Large Cytosolic Proteases for In Vitro Assays: 20S and 26S Proteasomes

Tobias HainHendrik BergerHansjörg SchildStefan Tenzer

subject

Differential centrifugationCytosolProteasesProteasomebiologyBiochemistryAntigen processingChemistrybiology.proteinMajor histocompatibility complexPolyacrylamide gel electrophoresisAmmonium sulfate precipitation

description

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.

https://doi.org/10.1007/978-1-62703-218-6_1