Powered by Boston Biochem: Products for Proteasome-related Research

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The Ubiquitin Proteasome system is essential for cellular protein homeostasis. Abnormal functioning contributes to a number of human pathologies including Parkinson’s Disease, various cancers, Alzheimer’s Disease, and ALS. Finally, inhibiting proteasome activity with small molecules such as Bortezomib and Carfilzomib has proven efficacious in the treatment of multiple myeloma.

• The widest selection of purified proteasomes
• Unsurpassed purity and functional characterization
• Fluorogenic substrates for activity testing
• β-Subunit specific probes

The Widest Selection of Purified Proteasomes

Unsurpassed Purity and Functional Characterization

Proteasome Purity Highlighted with SDS-PAGE. From 1-8 µg of human i20S proteasome (Catalog # E-370) was visualized by reducing SDS-PAGE and Colloidal Coomassie Blue staining. The 20S subunits are seen in the 25-35 kDa range, while HSP90, a tightly associated chaperone present in immunoproteasome obtained from PBMCs is seen on top.		Proteasome Activity Highlighted Using a Fluorogenic Substrate. Human 26S proteasome Catalog # E-365) was separated using a 5% native, non-reducing PAGE gel then analyzed for activity by soaking in LLVY-AMC substrate. Left panel: LLVY-AMC assay done the presence of 0.035% SDS. Right panel: LLVY-AMC assay done without 0.035% SDS. Digitometry values reveal that the purified proteasome consists of double-capped + single-capped species of 89%; uncapped (20S) proteasome was present at just 11%.

Fluorogenic Substrates for Activity Testing

Peptide-AMC conjugates are excellent substrates for monitoring proteasome activity, and may be used in either end-point or continuous monitoring (kinetic) formats. Depending on the peptide sequence, some substrates may be used with either constitutive or immunoproteasomes, while others show a marked difference in hydrolysis rates between the two proteasome types.

Proteasome Activity Measured using a Fluorogenic Subtrate. For the human 20S constitutive proteasome (left panel) and the human 20S immunoproteasome (right panel) activity was determined using the LLVY-AMC Fluorogenic Substrate (Catalog # S-280). This substrate is efficiently hydrolyzed by both proteasome types.

Coming soon: β-Subunit Specific Probes

In collaboration with Hermen Overkleeft and colleagues, Boston Biochem will soon offer a panel of tagged and fluorescent probes for labeling active β-subunits of the proteasome. These compounds are useful in many in vitro applications, including:

• Determining levels of constitutive vs immunoproteasome activity
• Following induction of immunoproteasome activity after interferon treatment
• Monitoring inhibition of β-subunit after treating cells with proteasome inhibitors

Labeling of Active β Subunits. Staining profile for Raji and HEK293 cells (left panel). The three color probes indicate the presence of consitutive β subunits 2c, 1c, and 5c (green, blue, and red, respectively) in cell lysates from HEK cells, indicating that these cells only express constitutive proteasomes. The Raji cell lysates show the presence of three additional bands that correlate to the three β subunits of the immunoproteasome as well. Right Panel: IFN-γ treatment induces immunoproteasome activity. RPMI cells express both constitutive and immunoproteasome activities, with the latter being upregulated following 24 hours of treatment with IFN-γ. HeLa cells lack appreciable immunoproteasome activity in the absence of IFN-γ, but the β1i, β2i, and β5i subunits are clearly upregulated. (Figures adapted from de Bruin G. et al. (2015) Angew. Chem. Int. Ed. 54:1.