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
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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
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