20S Proteasome Activity Assay;

20S Proteasome Activity Assay

Chemicon (Millipore)

Introduction:

The ubiquitin-proteasome pathway is the major proteolytic system in the cytosol of eukaryotic cells, where it catalyzes the selective degradation of short-lived regulatory proteins and the rapid elimination of proteins with abnormal conformation (Hershko & Ciechanover 1998; Hochstrasser 1996). The critical protease in this pathway is the 26S proteasome, an ATP-dependent proteolytic complex, which is formed by the association of the barrel-shaped 20S proteasome (700-kDa) and two 19S (700-kDa) regulatory complexes (Baumeister et al. 1998; Coux et al. 1996). The 20S Proteasome, catalytic core of the proteasome complex, is responsible for the breakdown of key proteins involved with apoptosis, DNA repair, endocytosis, and cell cycle control (Coux et al. 1996; Hoffman & Rechsteiner 1996; Hochstrasser 1995). The CHEMICON Proteasome Activity Assay Kit provides a quick, efficient and sensitive system for evaluation of proteasome activity in cell lysates or inhibitor screening.

Test Principle:

Chemicon's Proteasome Activity Assay Kit provides a simple and convenient means for assaying proteasome activity that recognize the substrate LLVY (Meng et al. 1999). The assay is based on detection of the fluorophore 7-Amino-4-methylcoumarin (AMC) after cleavage from the labeled substrate LLVY-AMC. Absorption=351nm. Emission=430nm. The free AMC fluorescence can be quantified using a 380/460 nm filter set in a fluorometer.

A proteasome inhibitor, Lactacystin, is included as a test inhibitor for screening purpose. Lactacystin is a microbial natural product and the most specific and potent inhibitor of proteasomes currently known (Fenteany & Schreiber 1998).

Application:

The Proteasome Activity Assay Kit is a relatively quick method for detection of intracellular proteasome activity. Testing of purified proteasome enzyme, in vitro inhibitor screening and the study of proteasome regulation can be performed with this assay.

The CHEMICON Proteasome Activity Assay Kit is intended for research use only, not for diagnostic or therapeutic applications.

Activity Assay

Lysis buffer: 50 mM HEPES (pH 7.5), 5 mM EDTA, 150 mM NaCl and 1% Triton X-100. Addition of 2 mM ATP to the lysate will improve the recovery of intact 26S proteasome.

Also see: Hoffman, L. 1992 at http://www.jbc.org/cgi/reprint/267/31/22362.pdf for additional methods for isolation.

Preparation of the proteasome sample can be done by many different methods. This paper in JBC is a good reference, http://www.jbc.org/cgi/reprint/267/31/22362.pdf, however the method unfortunately requires 100,000g spins and DEAE columns but it does provide very, very clean and active proteasomes,

An easier yet more crude preparation is typically done for most studies.

The cell lysis buffer is, 50 mM HEPES (pH 7.5), 5 mM EDTA, 150 mM NaCl and 1% Triton X-100. Addition of 2 mM ATP to the lysate will improve the recovery of intact 26S proteasome. The addition of ATP is optional.

Cell Extraction
Note: This protocol has been successfully applied to several cell lines.
Users should optimize the cell extraction procedure for their own applications.

1. Collect cells in PBS by centrifugation (non-adherent cells) or scraping from culture flasks (adherent cells).

2. Wash cells twice with cold PBS.

3. Remove and discard the supernatant and collect the cell pellet. At this point the cell pellet can be frozen at -80°C and lysed at a later date.

4. Lyse the cell pellet in 0.5 mL lysis buffer for 30 minutes, on ice, with vortexing at 10-minute intervals.

*To get a rough idea you could adjust the cell concentration to around 2 x 10⁷ cells/mL. Resulting protein concentration of the cell lysate should be 2-4 mg/mL using this lysis buffer.
**The volume of cell lysis buffer depends on the cell line, the cell number in cell pellet and the amount of poly-ubiquitinated protein. For example, 1 x 10⁷ MCF-7 cells can be extracted in 0.5 mL of cell lysis buffer.

5. Transfer the lysate to microcentrifuge tubes and centrifuge at 15,000 rpm for 15 minutes at 4°C.

6. Aliquot the clear extract to clean microcentrifuge tubes. These cell lysates are ready for assay.
The cell lysate can be stored at -80°C. Avoid multiple freeze/thaw cycles. After thaw the cell lysate, Centrifuge at 15,000 rpm for 15 minutes at 4°C again since the cell lysate should be clear of any sediments or particulate matter.

Isolation of 20S core: Additional info: The crude protocol will provide 20S material, not exclusively but the 20S will be present as it is the core of the 26S particle .

Lliterature references abound for isolation of 20S proteasome from various tissues including for example, skeletal muscle. The isolation is done essentially the same way as the crude method but the spins are much higher, with the final spin being 100,000 x g for six hours, after which the pellet is resuspended and pooled. A good general reference is the following, Hayter JR et al, 2005, Mol. Cell Proteomics, PMID:15965267

http://www.mcponline.org/cgi/reprint/M400138-MCP200v1.pdf.

"Purification of the 20S proteasome: The purification protocol was based upon previously published methods, adapted for purification of the complex from large or smaller quantities of tissue [see reference]. Chick skeletal muscle was mechanically homogenised using a domestic food processor in 1.5 volumes of buffer A (20mM Tris, 20mM KCl, 10mM magnesium acetate, 2mM DTT, 10% glycerol pH 7.6). The soluble protein fraction was isolated by centrifugation at 30,000g for 30min at 4°C.

The pellet was discarded and the supernatant fraction was centrifuged again at 100,000g for 6h. The supernatant fraction was again discarded and the pellet was washed carefully in fresh buffer, which was also discarded. The washed pellets were resuspended in 1ml of buffer A (per pellet), pooled and stored at -20°C in 1.5ml aliquots.

Mammals

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Signal Transduction Assays

• 20S Proteasome Positive Control (Part No. 90205): One vial containing 20S Proteasome Positive Control in 20 mM Tris, pH 7.2 and 1 mM DTT.
• 10X Assay Buffer (Part No. 90209): One 1.5 mL vial.
• Proteasome Substrate (Suc-LLVY-AMC) (Part No. 90206): One vial contains 500 μg peptide substrate.
• Lactacystin (20S Proteasome Inhibitor) (Part No. 90208): One vial contains 9.4 μg inhibitor.
• AMC Standard (Part No. 90207): One vial contains 2.2 μg AMC standard.

Store kit materials (as provided) at -20°C up to their expiration date.

• PSMA1
• MGC22853
• MGC14751
• PROS30
• MGC1667
• HC2
• MGC21459
• PSC2
• MGC14542
• PROS-30
• MGC23915
• NU
• MGC14575
• EC 3.4.25.1

· 96-well fluorometer plate

· Adjustable volume pipettor with disposable tips

· 37°C incubator

· DMSO

· Fluorometer with a 380/460nm filter set