Applications
- Capture of ubiquitylated proteins from cell lines, tissues and organs followed by Western blot detection.
- Immunoprecipitation of ubiquitylated proteins of interest. This application requires the elution of captured proteins, dialysis and immunoprecipitation with a specific antibody recognizing a protein of interest that are not provided in this kit.
Specificities
UBI trap-B is not crosslinked to agarose beads but preserved in a lyophilized state for long term storage. UBI trap-B is reconstituted with a specific buffer just before use and mixed with glutathione agarose beads provided in the kit. Washing and elution buffers are also included. Negative control GST is provided for a single control reaction. Captured proteins have to be boiled to be analyzed by Western blot. Since UBI traps are not crosslinked to agarose, these will be released during the boiling. Avoid using antibodies for Western blot analysis that have been generated with a GST fusion with the protein of interest, otherwise GST protein will be detected. If no alternative antibodies are available, we advise to use UBI traps tagged with biotin.
Characteristics
Tag: GST
Purity: > 95% by RP-HPLC and SDS-PAGE
Molecular Weight: 55,647 kDa
Physical State: Solid (powder)
Quantity: 200 µg
Solubility: >30 mg/mL
Concentration: Variable
Storage: RT but once reconstituted store at -80°C and avoid freeze/thaw cycles
Benefits
Higher affinity for polyubiquitin chains than a single UBD
Avoid overexpression of tagged ubiquitin for pull downs
Protect poly-ubiquitin chains without inhibitors specific to the proteasome or DUBs activity
Replace ubiquitin specific antibodies for enrichment of poly-ubiquitylated proteins
KIT UBI trap-B has been developed to facilitate the use, long-term stability and transport of all components required to capture ubiquitylated proteins using agarose beads.
Examples of use
UBI traps can be used to detect ubiquitylated cellular factors implicated in the regulation of signaling pathways regulating inflammation, oncogenesis, neurodegenerative diseases and multiple infections (4-8). The ubiquitylated status of these factors can be followed in response to a treatment and determine if cells are responding well (9-10). The ubiquitylation of crucial cellular factors can also be followed after infection with various microorganisms (11). Among other examples. A UBI traps step by step protocol has also been reported (12).
Publications
(1) Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. Hjerpe R, Aillet F, Lopitz-Otsoa F, Lang V, England P, Rodriguez MS.EMBO Rep. 2009 Nov;10(11):1250-8.
(2) Polyubiquitin-sensor proteins reveal localization and linkage-type dependence of cellular ubiquitin signaling. Sims JJ, Scavone F, Cooper EM, Kane LA, Youle RJ, Boeke JD, Cohen RE.Nat Methods. 2012 Feb 5;9(3):303-9.
(3) Using Ubiquitin Binders to Decipher the Ubiquitin Code. Mattern M, Sutherland J, Kadimisetty K, Barrio R, Rodriguez MS.Trends Biochem Sci. 2019 Jul;44(7):599-615.
(4)Role of monoubiquitylation on the control of IκBα degradation and NF-κB activity. Da Silva-Ferrada E, Torres-Ramos M, Aillet F, Campagna M, Matute C, Rivas C, Rodríguez MS, Lang V. PLoS One. 2011;6(10):e25397.
(5) Tetramerization-defects of p53 result in aberrant ubiquitylation and transcriptional activity. Lang V, Pallara C, Zabala A, Lobato-Gil S, Lopitz-Otsoa F, Farrás R, Hjerpe R, Torres-Ramos M, Zabaleta L, Blattner C, Hay RT, Barrio R, Carracedo A, Fernandez-Recio J, Rodríguez MS, Aillet F. Mol Oncol. 2014 Jul;8(5):1026-42.
(6) Inhibition of the proteasome and proteaphagy enhances apoptosis in FLT3-ITD-driven acute myeloid leukemia. Lopez-Reyes RG, Quinet G, Gonzalez-Santamarta M, Larrue C, Sarry JE, Rodriguez MS. FEBS Open Bio. 2021 Jan;11(1):48-60.
(7) Analysis of defective protein ubiquitylation associated to adriamycin resistant cells. Lang V, Aillet F, Xolalpa W, Serna S, Ceccato L, Lopez-Reyes RG, Lopez-Mato MP, Januchowski R, Reichardt NC, Rodriguez MS. Cell Cycle. 2017;16(24):2337-2344.
(8) Activation of AHR mediates the ubiquitination and proteasome degradation of c-Fos through the induction of Ubcm4 gene expression. Mejía-García A, González-Barbosa E, Martínez-Guzmán C, Torres-Ramos MA, Rodríguez MS, Guzmán-León S, Elizondo G. Toxicology. 2015 Nov 4;337:47-57.
(9) Activation of aryl hydrocarbon receptor regulates the LPS/IFNgamma-induced inflammatory response by inducing ubiquitin-proteosomal and lysosomal degradation of RelA/p65. Domínguez-Acosta O, Vega L, Estrada-Muñiz E, Rodríguez MS, Gonzalez FJ, Elizondo G. Biochem Pharmacol. 2018 Sep;155:141-149.
(10) Efficient monitoring of protein ubiquitylation levels using TUBEs-based microarrays. Serna S, Xolalpa W, Lang V, Aillet F, England P, Reichardt N, Rodriguez MS. FEBS Lett. 2016 Aug;590(16):2748-56.
14;14:200.
(11) New insights into host-parasite ubiquitin proteome dynamics in P. falciparum infected red blood cells using a TUBEs-MS approach. Mata-Cantero L, Azkargorta M, Aillet F, Xolalpa W, LaFuente MJ, Elortza F, Carvalho AS, Martin-Plaza J, Matthiesen R, Rodriguez MS. J Proteomics. 2016 Apr 29;139:45-59.
(12) Isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. Aillet F, Lopitz-Otsoa F, Hjerpe R, Torres-Ramos M, Lang V, Rodríguez MS.Methods Mol Biol. 2012;832:173-83.
