For isolation and enrichment of ubiquitinylated proteins.The UbiQapture™-Q Kit is an efficient tool for the selective isolation of ubiquitinylated proteins. The Kit facilitates the isolation of both mono- and poly-ubiquitinylated proteins (independent of lysine residue chain linkage) from cell extracts, tissue lysates and
in vitro assay solutions through use of a high-binding affinity matrix. Captured proteins can be analyzed by Western blotting using the highly sensitive ubiquitin-conjugate specific antibody provided, using antibodies to specific proteins of interest, or eluted from the matrix for subsequent biochemical characterization.
The UbiQapture™-Q matrix supplied with the Kit has superior binding characteristics compared to other commercially available matrices and is compatible with a wide range of lysate buffers and cell/tissue samples from a variety of species.Suggested uses: Isolation and detection of ubiquitinylated protein conjugates from a specific cell/tissue lysate, Capture and analysis of specific ubiquitinylated protein conjugates of interest from particular cell/tissue lysates, Release of free proteins in their active/native form by cleavage of ubiquitin/ubiquitin chains from the UbiQapture™-Q matrix using a deubiquitinylating enzyme, Release of ubiquitinylated proteins in their active/native form by elution from the UbiQapture™-Q matrix using high salt buffer.
Figure: Western blot analysis of ubiquitin enrichment of lysate derived ubiquitinylated proteins. Ubiquitin enrichment experiment set-up and run as described in “Capture/enrichment of ubiquitinylated proteins”. Ubiquitin-protein conjugates present in starting material, unbound fraction and elution fraction were detected by western blotting as described in “Analysis by western blotting”, using the provided ubiquitin-conjugate specific HRP-linked antibody (Prod. No. BML-UW0150) at a dilution of 1:1000 dilution. Capture of Ub-protein conjugates from Control ubiquitinylated-protein lysate (Prod. No. BML-UW0130). Key: SM = Starting Material, UF = Unbound Fraction and EL = Elution Fraction.
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Product Specification
| Quantity: | Provides sufficient material for 20 x 20μl reactions. |
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| Kit/Set Contains: |
UbiQapture™-Q matrix (Prod. No. BML-UW0125) 0.5 ml settled resin provided; 50% suspension.
Control ubiquitinylated-protein lysate (Ub-lysate) (Prod. No. BML-UW0130) 500 µg (100µl), 5mg/ml in 50mM HEPES, pH 7.6, 1mM DTT.
Ubiquitin-conjugates specific HRP-linked antibody solutions (Prod. No. BML-KW0150) 25µl; HRP-linked antibody for use with ECL western blotting detection reagents.
0.5ml screw capped tube pack (Prod. No. BML-UW0135) 25 tubes. |
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| Application: | 1. Isolation and detection of the full range of ubiquitinylated protein conjugates (mono-/multi-/poly-ubiquitin modified, lysine linkage independent) from specific cell/tissue lysates of interest, especially low abundance (regulatory) proteins involved in the ubiquitin-proteasome pathway.
2. Capture and analysis of specific ubiquitinylated protein conjugates of interest from particular cell/tissue lysates.
3. Purification/pull down of ubiquitinylated proteins from cell free in vitro assays; for example to isolate Ub-p53 species from ubiquitinylation assays mediated by HeLa S100 lysate (Prod. No. BML-SW8750).
4. Release of free proteins in their active/native form by cleavage of ubiquitin/ubiquitin chains from the UbiQapture-Q matrix using deubiquitinylating enzymes such as USP2 (Prod. No. BML-UW9850).
5. Release of ubiquitinylated proteins in their active/native form by elution from the UbiQapture-Q matrix using, for example, high salt buffer.
Note: Protocol provided covers applications 1-3. Assay set-up can be readily modified for alternative applications by inclusion, omission or
substitution of specific components.
Kit is not suitable for use with purified proteins |
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| Long Term Storage: | -80°C |
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| Use/Stability: | Upon receipt, the UbiQapture™-Q matrix (Prod. No. BML-UW0125) should be strored at +4°C. All other biochemical components should be stored at -80°C. |
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| Background / Technical Information: | Please click here for the comprehensive product datasheet. |
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General Literature References
BAX/BAK-independent mitoptosis during cell death induced by proteasome inhibition?: E. Lomonosova, et al.; Mol. Cancer Res.
7, 1268 (2009),
Abstract;
Homologous recombination but not nucleotide excision repair plays a pivotal role in tolerance of DNA-protein cross-links in mammalian cells: T. Nakano, et al.; J. Biol. Chem.
284, 27065 (2009),
Abstract;
Diverse polyubiquitin interaction properties of ubiquitin-associated domains: S. Raasi et al.; Nat. Struct. Mol. Biol.
12, 708 (2005),
Abstract;
Ubiquitin-binding domains: L. Hicke et al.; Nat. Rev. Mol. Cell. Biol.
6, 610 (2005),
Abstract;
Polyubiquitin chains: polymeric protein signals: C.M. Pickart et al.; Curr. Opin. Chem. Biol.
8, 610 (2004),
Abstract;
Mechanisms underlying ubiquitination: C.M. Pickart et al.; Annu. Rev. Biochem.
70, 503 (2001),
Abstract;
Protein regulation by monoubiquitin: L. Hicke et al.; Nat. Rev. Mol. Cell. Biol.
2, 195 (2001),
Abstract;
The ubiquitin-proteasome system and endocytosis: G.J. Strous et al.; J. Cell. Sci.
112 , 1417 (1999),
Abstract;
The ubiquitin system: A. Hershko et al.; Annu. Rev. Biochem.
67, 425 (1998),
Abstract;
Pathways of ubiquitin conjugation: A.L. Haas et al.; Faseb. J.
Faseb J, 1257 (1997),
Abstract;
United States
