PMC

In-gel peptidase acitivity assay to measure 20S and 26S proteasome activity. In-gel peptidase activity assay was performed as described in Basic Protocol 4. (A) A 4% native polyacrylamide gel incubated with the developing buffer without SDS. (B) A 4% native polyacrylamide gel incubated with the developing buffer containing 0.02% SDS. (A) and (B) are visualizations of same samples before and after adding 0.02% SDS. Left lane: 0.10 μg of 20S proteasome (CP). Right lane: 0.37 μg of 26S proteasome (RP₂CP and RPCP). (C) A 4% native gel stained with Gelcode Blue (Coomassie Blue). Left lane: 0.30 μg 20S proteasome. Right lane: 1.1 μg 26S proteasome. Proteasome species RP₂CP, RPCP and CP are labeled in the figure. In Figure 2A and Figure 2B, there are one or two very weakly stained bands with chymotryptic activity detected in between RP₂CP and RPCP, and one or two additional species in between RPCP and CP. The exact compositions of these complexes were not examined. They presumably contain known substoichiometric proteasome-binding proteins such as Hul5, Ubp6, Ecm29 or Blm10, in addition to the RP and CP.

Purified yeast 20S CP and 26S proteasome subunits resolved by electrophoresis through a 12% SDS-polyacrylamide gel. The 20S CP and 26S proteasomes were purified as described in Basic Protocol 3 and Basic Protocol 1, respectively. The gel was stained with Gelcode Blue at room temperature for one hour. Lane 1 contains molecular weight markers. Lanes 2 to 5 are BSA standards with, respectively, 750 ng, 500 ng, 250 ng and 125 ng BSA loaded per lane. Lane 6 is loaded with 2.6 μg of purified 20S proteasome (CP). Lane 7 is loaded with 7.8 μg of purified 26S proteasomes. The 20S proteasome is purified through Pre1 (β4)-3xFLAG, which has a molecular weight shift from 22.5 kDa to 25.3 kDa. It migrates together with other subunits at around 26 kDa, shown in Lane 6 as a dark band that poorly separated. The 26S proteasome is purified through Rpn11–3xFLAG, which has a molecular weight shift from 34.4 kDa to 37.2 kDa. It migrates together with Rpn8 and Rpn9 and can not be distinguished in a 12% SDS-PAGE gel as shown in Lane7. Rpn3 (molecular weight 60377.3 Da) and Pre10 (α7, molecular weight 31521.3 Da) are labeled as our choice of the subunits for quantification.