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Proteasome 26S degradation kit

 
BML-PW8950-0001 96 wells 291.00 USD
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Replaces Prod. #: ALX-850-231

This kit contains a highly purified, human erythrocyte derived, preparation of 26S proteasomes useful for carrying out in vitro protein degradation studies with suitably ubiquitinylated protein substrates. The preparation consists of a high purity mixture of 26S proteasomes singly (26S) and doubly (30S) capped with 19S regulatory subunit complexes in the ratio of 40% single cap : 60% double cap at the time of preparation. Additional kit components include ATP for proteasomal activation.

Product Specification

Quantity:1 plate, 1x96 wells
 
Use/Stability:Care must be exercised in the handling and storage of the 26S proteasome to ensure that its activity is not comprimised. The complex should be stored at -80°C. When ready for use it should be thawed by standing on ice (i.e. not thawed rapidly!). For those wishing to measure and use the enzymatic activity of 26S proteasome it should be used immediately after it is thawed since the enzyme complex is labile after thawing. After dissociation of the complex, the 20S proteasome activity is relatively stable.
 
Shipping:Shipped on Dry Ice
 
Long Term Storage:-80°C
 
Kit/Set Contains:MgATP (Prod. No. BML-EW9805-0125, 0.1M MgATP, 125 μl)
26S Proteasome (Prod. No. BML-PW9310-0050, 50μg in 10mM TRIS, containing 25mM potassium chloride, 1.1mM magnesium chloride, 0.1mM ethylenediaminetetraacetic acid, 1mM dithiothreitol, 1mM sodium azide, 2mM ATP, pH 7.0, and 35% glycerol)
Radio-labelled ubiquitinylated protein conjugate [NOT SUPPLIED]
 
Protocol:Please Note: The following are general guidelines. Individual results may vary with different conditions and substrates.
Components (quantity/final concentration):
  • MgATP=2 mM
  • 26S proteasome=5-10nM depending upon conditions
  • Radio-labelled ubiquitinylated protein conjugate=50-100nM (5000-15000cpm)
Procedure for monitoring TCA soluble cpm:
1. Add all components, i.e. conjugates, MgATP, 26S proteasome.
2. Incubate reaction mixture at 37C for 0-90 minutes.
3. At desired time points, take 10μL of reaction mixture and add to quenching buffer containing 600μL of 10% trichloroacetic acid (TCA) and 200μL 5% bovine serum albumin as a carrier.
4. Vortex and place on ice for 15 minutes.
5. Centrifuge at 4C for 10 minutes in microcentrifuge at 14000rpm.
6. Count 650μL of supernatant in scintillation counter.
7. Plot increase in TCA soluble counts versus time.
 

Product Literature References

Reconstitution of hybrid proteasomes from purified PA700-20 S complexes and PA28alphabeta activator: ultrastructure and peptidase activities: F. Kopp et al.; J. Mol. Biol. 313, 465 (2001), Abstract;
A multicomponent system that degrades proteins conjugated to ubiquitin. Resolution of factors and evidence for ATP-dependent complex formation: D. Ganoth et al.; J. Biol. Chem. 263, 12412 (1988), Abstract;
The protein substrate binding site of the ubiquitin-protein ligase system: A. Hershko et al.; J. Biol. Chem. 261, 11992 (1986), Abstract;
ATP-dependent degradation of ubiquitin-protein conjugates: A. Hershko et al.; PNAS 81, 1619 (1984), Abstract;
Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown: A. Hershko et al.; J. Biol. Chem. 258, 8206 (1983), Abstract;

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