539176 Proteasome Isolation Kit, Human

539176
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      Overview

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      539176-1KIT
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          Description
          OverviewA rapid method for the isolation of biologically active proteasomes using affinity matrix beads comprised of a GST-fusion protein containing an ubiquitin-like domain (UbL) bound to GST-agarose. The proteasome subunit proteins can be identified by loading the beads directly onto an SDS-PAGE gel and immunoblotting with subunit specific antibodies. Alternatively, proteasome bound to beads can be used in proteolytic assays using proteasome substrates.

          Utilize Ubiquitin-like domain (Ubl) bound to GST-agarose
          Rapidly isolate biologically active proteasomes
          Affinity mature beads can be directly loaded onto SDS-PAGE gels for proteasome subunit identification
          Proteasome-bound beads are compatible with proteolytic assays using proteasome substrates

          Catalogue Number539176
          Brand Family Calbiochem®
          Application Data
          Total protein extracts were applied to control and proteasome-binding beads, and incubated for 4 h at 4°C. The matrix was washed three times with Buffer A, suspended in SDS sample buffer, separated in a 10% SDS-polyacrylamide gel, and electroblotted to nitrocellulose. The filter was incubated with antibodies against Rpt1, a subunit in the 19S regulatory particle of the proteasome. The first lane shows the recovery of the proteasome subunit Rpt1 with the proteasome-binding GST-UbL beads. The second lane is a mock-reaction (using GST), while the third lane contains 5% of the input lysate. Similar samples were incubated in reaction buffer to measure proteasome-specific proteolytic activity. The hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC was readily detected in the GST-UbL beads, while the no activity was observed with the GST beads.
          Materials Required but Not Delivered Lysis buffer: 50 mM HEPES (pH 7.5), 5 mM EDTA, 150 mM NaCl and 1% Triton® X-100 detergent. Addition of 2 mM ATP to the lysate will improve the recovery of intact 26S proteasome.
          Wash Buffer (Buffer A): Lysis buffer with protease inhibitors such as Antipain, aprotinin, chymostatin, leupeptin, and pepstatin. Alternatively, a pre-made protease inhibitor cocktail such as Cat. No. 539134 can be used.
          Reaction Buffer: 25 mM Hepes, 500 mM EDTA, pH 7.6
          Gel Loading Buffer: 250 mM Tris, HCl, pH 6.8; 4% SDS; 10% β-mercaptoethanol; 20% glycerol; and bromophenol blue
          TBST: 10 mM Tris-HCl, 150 mM NaCl, 0.1% TWEEN®-20 detergent
          References
          ReferencesChen, L., and Madura, K. 2002. Mol. Cell Biol. 22, 4902.
          Chen, L., et al. 2001. EMBO Rep. 2, 933.
          Schauber, C., et al. 1998. Nature 391, 715.
          Product Information
          DeclarationSold under license of U.S. Patent 6,294,363.
          Format1.0 ml bead suspension
          Kit containsUbL Beads Sufficient to process 12.5 to 25 mg of Lysate, Control Glutathione-Agarose Beads, Control Lysate, and a user protocol.
          Applications
          Biological Information
          Assay time3-5 h
          Sample TypeCell extracts
          Species Reactivity
          • Canine
          • Human
          • Mouse
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Storage and Shipping Information
          Ship Code Blue Ice Only
          Toxicity Standard Handling
          Storage +2°C to +8°C
          Storage ConditionsUpon arrival store entire kit contents at 4°C.
          Do not freeze Yes
          Packaging Information
          Transport Information
          Supplemental Information
          Kit containsUbL Beads Sufficient to process 12.5 to 25 mg of Lysate, Control Glutathione-Agarose Beads, Control Lysate, and a user protocol.
          Specifications

          Documentation

          Proteasome Isolation Kit, Human SDS

          Title

          Safety Data Sheet (SDS) 

          Proteasome Isolation Kit, Human Certificates of Analysis

          TitleLot Number
          539176

          References

          Reference overview
          Chen, L., and Madura, K. 2002. Mol. Cell Biol. 22, 4902.
          Chen, L., et al. 2001. EMBO Rep. 2, 933.
          Schauber, C., et al. 1998. Nature 391, 715.

          Citations

          Title
        • Yolanda Fernandez, et al. (2006) Chemical Blockage of the Proteasome Inhibitory Function of Bortezomib: Impact on Tumor Cell Death. Journal of Biological Chemistry 281, 1107-1118.
        • User Protocol

          Revision18-August-2010 RFH
          Format1.0 ml bead suspension
          Speciescanine, human, mouse
          StorageUpon arrival store entire kit contents at 4°C.
          BackgroundProteasomes are large multi-subunit protease complexes, localized in the nucleus and cytosol that selectively degrade intracellular proteins. They play a major role in the degradation of many proteins involved in cell cycling, proliferation, and apoptosis. The ubiquitin-proteasome pathway degrades a vast majority of short-lived proteins. A protein marked for degradation is covalently attached to multiple molecules of ubiquitin (Ubq), which escorts it for rapid hydrolysis to the multi-component enzymatic complex known as the 26S proteasome. The proteolytic core of this complex, the 20S proteasome, contains multiple peptidase activities and functions as the catalytic machine. This core is composed of 28 subunits arranged in four heptameric, tightly stacked rings (α7, β7, β7, α7) to form a cylindrical structure. The α-subunits make up the two outer, and the β-subunits the two inner, rings of the stack. The entrance of substrate proteins to the active site of the complex is guarded by the α-subunits that only allow access for unfolded and extended polypeptides. The proteolytic activity is confined to the β-subunits. The regulatory unit of the 26S proteasome is known as the 19S (PA 700) particle, and consists of at least 15 subunits that include ATPases, a de-ubiquitinating enzyme, and polyubiquitin-binding subunits. ATPases function to continuously supply energy for selective degradation of target proteins. Energy is required for unfolding of proteins to allow them to penetrate the channel of α-and β-rings of the 20S proteasome. PA28 (11S regulator), a ring-shaped particle that associates with the 19S unit at both ends of the 20S proteasome, functions as an activator protein. It is composed of two homologous proteins, PA28α and PA28β and is reported to stimulate peptidase activities without affecting the degradation of large protein substrates. Rad23 belongs to a large family of proteins bearing ubiquitin-like domains that have been shown to interact with the 26S proteasome. Rad23 or its ubiquitin-like domain (UbLR23) fused to GST was shown to pull down 20S proteasome subunits containing chymotrypsin-like proteolytic activity. Two human homologues of Rad23 containing the ubiquitin-like domains have been identified, HHR23-A and HHR23-B. The human Proteasome Isolation Kit contains the ubiquitin-like domain of HHR23-B (UbLHRB) fused to GST and bound to GST-Agarose. This kit can be used to isolate proteasome subunits from cell extracts to study their function and interactions with other proteins. The proteasome subunits can be identified by loading the beads directly onto an SDS-PAGE gel and immunoblotting with subunit specific antibodies. Alternatively, proteasome bound to the beads can be used in proteolytic assays using proteasome substrates.
          Materials provided(Quantities sufficient for 25 affinity isolation assays)

          • Proteasome Affinity Beads (Kit Component No. KP31240): 20% Suspension of affinity beads in PBS, 0.2% NaN₃, pH 7.1. 1.0 ml.
          • Control Beads (Kit Component No. KP31241): Suspension of control beads in PBS, 0.2% NaN₃, pH 7.1. 200 µl.
          • Control Lysate (Kit Component No. KP31242): Suspension of protein extract in 1.5 M Tris-HCl, 10% glycerol, 5% β-mercaptoethanol, 2% SDS, 0.02% bromophenol blue, pH 6.8. Useful as an immunoblot standard. 60 µl.
          Materials Required but not provided Lysis buffer: 50 mM HEPES (pH 7.5), 5 mM EDTA, 150 mM NaCl and 1% Triton® X-100 detergent. Addition of 2 mM ATP to the lysate will improve the recovery of intact 26S proteasome.
          Wash Buffer (Buffer A): Lysis buffer with protease inhibitors such as Antipain, aprotinin, chymostatin, leupeptin, and pepstatin. Alternatively, a pre-made protease inhibitor cocktail such as Cat. No. 539134 can be used.
          Reaction Buffer: 25 mM Hepes, 500 mM EDTA, pH 7.6
          Gel Loading Buffer: 250 mM Tris, HCl, pH 6.8; 4% SDS; 10% β-mercaptoethanol; 20% glycerol; and bromophenol blue
          TBST: 10 mM Tris-HCl, 150 mM NaCl, 0.1% TWEEN®-20 detergent
          Detailed protocolProtocol for using the proteasome affinity matrix

          1. Resuspend the matrix by gentle inversion, until the beads are completely unpacked.
          2. Use a large bore pipet tip (prepared by cutting off the terminal 3 mm with a razor blade) to remove ~40 µl of the affinity bead suspension. There is no need to wash the beads.
          3. Add the beads directly to 0.5 mg-1.0 mg of cell lysate, at a concentration of ~1 mg/ml. The protein sample should be clear of any sediments or particulate matter, since this material will be recovered with the beads through subsequent washes.

          *Cell lysates can be prepared using Lysis buffer pre-cooled to 4°C. Cells can be further disrupted by sonication, hydrostatic pressure, glass-bead agitation, or with osmotic destabilizers.

          4. Incubate at 4°C for 2-4 h with constant mixing to keep the affinity beads well suspended. Avoid aeration or vigorous mixing.
          5. Remove supernatant after centrifugation for 5 s at 4°C in a microfuge (~1000 X g), and resuspend in 1 ml of Wash buffer pre-cooled to 4°C. Repeat three more times.
          6. Suspend the affinity matrix in 40 µl of 2X gel loading buffer (gel loading buffer should be at room temperature), and boil for 5 min.
          7. Centrifuge the material for 1 min at full speed in a microfuge (>10,000 X g), and apply the supernatant to an 8-12% SDS-PAGE.
          8. For immunoblotting, transfer the resolved proteins to nitrocellulose (preferably 0.2 µm), and stain with Ponceau S to confirm efficiency of transfer.

          To confirm that proteasome subunits are recovered:

          9. Wash the immunoblot 3 times with 1X TBST, using 100 ml per wash for 10 min each.
          10. Block the nitrocellulose membrane for 30 minutes on a rocking platform with TBST 5% milk solution for monoclonals and 10% for polyclonals at room temperature using about 1 ml per cm2 of membrane.
          11. Wash the nitrocellulose membrane 4 times in TBST for 10 min each on a rocking platform.
          12. Incubate the nitrocellulose membrane for 60 min on a rocking platform with a primary antibody specific to a component of the 19S particle of the proteasome diluted in TBST, 5% milk. If all the affinity binding steps (#3-5 above) are conducted in the presence of ATP, the recovery of the intact 26S proteasome (19S regulatory and 20S catalytic particles) will be improved.
          13. Wash the nitrocellulose membrane 4 times in TBST for 10 min each on a rocking platform.
          14. Incubate with secondary antibody enzyme conjugate diluted in 5% milk TBST for 60 min on a rocking platform.
          15. Wash the nitrocellulose membrane 4 times in TBST for 10 min each on a rocking platform.
          16. Develop with enhanced chemiluminescence to maximize detection.

          To confirm that the intact proteasome is active:

          17. The affinity beads containing purified proteasome (after the conclusion of step #5) can be washed and incubated in Reaction buffer containing a 10 µM fluorogenic substrate such as Suc-Leu-Leu-Val-Tyr-AMC (Cat. No. 539142) to measure the proteolytic activity of the catalytic particle.

          Figure 1: Sample Blot

          Total protein extracts were applied to control and proteasome-binding beads, and incubated for 4 h at 4°C. The matrix was washed three times with Buffer A, suspended in SDS sample buffer, separated in a 10% SDS-polyacrylamide gel, and electroblotted to nitrocellulose. The filter was incubated with antibodies against Rpt1, a subunit in the 19S regulatory particle of the proteasome. The first lane shows the recovery of the proteasome subunit Rpt1 with the proteasome-binding GST-UbL beads. The second lane is a mock-reaction (using GST), while the third lane contains 5% of the input lysate. Similar samples were incubated in reaction buffer to measure proteasome-specific proteolytic activity. The hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC was readily detected in the GST-UbL beads, while the no activity was observed with the GST beads.


          Cross-Reactivity

          This assay has also been tested and reported to work for canine and mouse tissues and cells.
          Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
          Triton® is a registered trademark of Dow Chemical Company
          Tween® is a registered trademark of ICI Americas, Inc.
          Interactive Pathways™ is a trademark of EMD Chemicals, Inc.