MultiDsk Ubiquitin Binding Protein Reagent

MultiDsk is a novel ubiquitin-binding protein reagent and is composed of an array of five UBA domains from the yeast ubiquitin-binding protein Dsk2, fused to GST. This novel reagent allows for the capture of ubiquitinated proteins from cell extracts and also provides a strong protective function, inhibiting de-ubiquitination.

Highlights:

  • Binds ubiquitinated substrates with unprecedented avidity (higher than GST-Dsk2 and TUBE-I)
  • GST-fusion allows for use with glutathione agarose beads as an affinity resin to study protein ubiquitination
  • Effectively protects ubiquitinated proteins from the action of de-ubiquitylating enzymes and the proteasome in crude cell extracts
  • Protective role at concentrations as low as 0.2 µM, making it suitable for use in large-scale extract experiments
  • Can be used to efficiently purify both mono- and poly-ubiquitinated proteins
  • Flexible linker between individual UBA domains allows free rotation of domains to accommodate multiple Ub chain topologies.
  • Highly stable: Can tolerate high salt and detergent conditions whilst still binding
  • Highly specific: Does not bind Ubiquitin-like Modifiers (UBLs) SUMO and NEDD8 

Ubiquitination is a highly diverse and complex post-translational modification for the regulation of protein function and stability. Studies of ubiquitination have, however, been hampered by its rapid reversal in cell extracts, for example through the action of de-ubiquitylating enzymes (DUBs). MultiDsk allows for the capture of pure, native, nontagged, ubiquitinated proteins from cell extracts whilst helping ensure the maintenance of the normal ubiquitination state via a strong protective function, inhibiting de-ubiquitination.

From a laboratory at Cancer Research Technology.

The Investigator's Annexe Part of The Investigator's Annexe program.

Catalog Number Product Size AVAILABILITY Price Qty
EW3001
MultiDsk Ubiquitin Binding Protein Reagent, 10ug
10ug (0.95mg/mL) In stock
Price: $279.00
Specifications
Product Type: Protein
Name: MultiDsk (array of 5 UBA domains)
Accession ID: P48510
Source: BL-21 (DE3) E. coli
Molecular Weight: 60 kDa including tags
Amino Acid Sequence: 5 tandem repeats of coding sequence for the yeast Dsk2 ubiquitin binding domain.
Fusion Tag(s): proprietary tag C-Terminus
Buffer: 25mM Tris-HCl pH7.5, 150mM NaCl, 50% glycerol, 2mM beta-mercapto-enthanol
Tested Applications: Isolation or protection of ubiquitinatedproteins in crude cell lysates
Concentration: 0.95mg/mL
Storage: 4C for up to 2 weeks (?20C or ?80C for long term storage)
Shipped: Dry ice
Data

Western Blot Analysis of Ubiquitin Capture and Protection

(A) MultiDsk binds ubiquilated proteins. 2 mg of yeast whole cell lysate was incubated with glutathione agarose beads. Differing amounts of GST protein, alone or asa mixture with GST-MultiDsk, were mixed with so that equal amounts of total protein and bead bed volumes were used in the comparison. Final concentration of MultiDsk protein in the pull-down is indicated. Western blot was performed using antiubiquitin antibody. (B) MultiDsk offers substantial protection against protein de-ubiquitination. Extract from strain W303 - expressing Myc-tagged ubiquitin was incubated with GST protein alone (negative control), 2mM n-ethylmaleimide [NEM] (positive control), or MultiDsk, as indicated. It was then left for 0, or 6 hours at 30?C, prior to Western blot analysis using anti-ubiquitin antibody to detectubiquitylated proteins.

Adapted from: Wilson MD, et al. PLoS One. 2012; 7 (10): e46398.

Comparison of Protection of Poly-ubiquitin Chains in Extract

Extract from strain SUB592 was incubated with equivalent amounts of GST, GST-Dsk2, commercially available TUBE-1, and MultiDsk and incubated at 30C for the indicated time. Total protein extracts were subject to Western blot and probed using anti-myc antibody.

Adapted from: Wilson MD, et al. PLoS One. 2012; 7 (10): e46398.

MultiDsks can be used to characterize the kinetics of ubiquitination of a specific protein species

(A) Exponentially growing yeast cells were either treated with 10 ?g/ml of 4-NQO for one hour, or left untreated, as indicated. Equal amounts of extracts were incubated with MultiDsk resin. Dilute Input extract (1%) and washes from the beads were also loaded. Proteins were analysed by Western blotting using anti-Rpb1 antibody, 4H8. (B) Exponentially growing yeast cells were either treated with 10 ?g/ml of 4-NQO for one hour, or left untreated, as indicated. Equal amounts of the extracts were incubated with agarose beads loaded with GST alone, GST-Dsk2 protein, commercial TUBE-1, or MultiDsk. Proteins were eluted via boiling in samplebuffer and subjected to Western blot analysis using the anti-Rpb1 antibody, 4H8 (upper panel). Ponceau S staining (lower panel) shows relative amounts of affinity proteins used. (C) Yeast cells were harvested at the indicated time after treatment with 4-NQO (10 ?g/ml), incubated with MultiDsk resin, and isolated proteins were analysed by Western blotting using either 4H8, or an anti-Def1 antibody, as indicated.

Adapted from: Wilson MD, et al. PLoS One. 2012; 7 (10): e46398.

Provider
From a laboratory at Cancer Research Technology.
References
  1. Ranes M, Boeing S, Wang Y, Wienholz F, Menoni H, Walker J, Encheva V, Chakravarty P, Mari PO, Stewart A, Giglia-Mari G, Snijders AP, Vermeulen W, Svejstrup JQ. A ubiquitylation site in Cockayne syndrome B required for repair of oxidative DNA damage, but not for transcription-coupled nucleotide excision repair. Nucleic Acids Res. 2016 Jun 20;44(11):5246-55.
  2. Wilson MD, Harreman M, Taschner M, Reid J, Walker J, Erdjument-Bromage H, Tempst P, Svejstrup JQ. Proteasome-mediated processing of Def1, a critical step in the cellular response to transcription stress. Cell. 2013 Aug 29;154(5):983-95.
  3. Wilson MD, Saponaro M, Leidl MA, Svejstrup JQ. MultiDsk: a ubiquitin-specific affinity. PLoS One. 2012; 7 (10): e46398.
  4. Liao C, Beveridge R, Hudson JJR, Parker JD, Chiang SC, Ray S, Ashour ME, Sudbery I, Dickman MJ, El-Khamisy SF. UCHL3 Regulates Topoisomerase-Induced Chromosomal Break Repair by Controlling TDP1 Proteostasis. Cell Rep. 2018 Jun 12;23(11):3352-3365. View Article

If you publish research with this product, please let us know so we can cite your paper.

 
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