Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Xu G, Paige JS, Jaffrey SR. Nat Biotechnol. 2010 Aug;28(8):868-73.
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A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Wagner SA, Beli P, Weinert BT, Nielsen ML, Cox J, Mann M, Choudhary C.Mol Cell Proteomics. 2011 Sep;10(10):M111.013284.
Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues. Wagner SA, Beli P, Weinert BT, Schölz C, Kelstrup CD, Young C, Nielsen ML, Olsen JV, Brakebusch C, Choudhary C.Mol Cell Proteomics. 2012 Dec;11(12):1578-85.
Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass. Povlsen LK, Beli P, Wagner SA, Poulsen SL, Sylvestersen KB, Poulsen JW, Nielsen ML, Bekker-Jensen S, Mailand N, Choudhary C. Nat Cell Biol. 2012 Oct;14(10):1089-98.
Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies. Swatek KN, Aumayr M, Pruneda JN, Visser LJ, Berryman S, Kueck AF, Geurink PP, Ovaa H, van Kuppeveld FJ, Tuthill TJ, Skern T, Komander D.Proc Natl Acad Sci USA. 2018 Mar 6;115(10):2371-2376
Insights into ubiquitin chain architecture using Ub-clipping. Swatek KN, Usher JL, Kueck AF, Gladkova C, Mevissen TE, Pruneda JN, Skern T, Komander D.Nature. 2019 Aug;572(7770):533-537.
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Monoclonal antibodies are produced by immunizing animals with a synthetic diglycine-modified histone peptide and established in hybridoma line GX41. Antibodies are purified by protein A and peptide affinity chromatography.
Each antibody batch is measured for its ability to bind to proteins containing diglycyl-lysine and to recover diglycyl-lysine-containing peptides from tryptic digests of HEK293 cells.
Cornell University has filed a use patent on proteomic methods that use the diglycyl-lysine antibody to identify ubiquitinated proteins. All commercial users should contact Cornell University for a use license.
Antibody recognizes lysine residues modified by diglycine, an adduct left at sites of ubiquitination after trypsin digest.
Figure 1: GX41 antibody recognizes the diglycyl-lysine moiety at ubiquitinated sites after trypsin digestion.
Figure 2: Specificity of anti-diglycyl-lysine (clone GX41) is verified by western blot analysis of beta-lactoglobulin, lysozyme, or rat brain lysate, in which the lysines were either unmodified (A), or modified with Boc-Gly-Gly (B) or Gly-Gly (C) adducts, respectively. As shown above, the GX41 exhibits no detectable reactivity with proteins unless the lysines are modified with Gly-Gly.
Figure 3: Equimolar amounts of the two peptides (GGDRVYIHPFHL and Ac-SYSMEHFRWGK*PV-NH2) were immunoprecipitated with immobilized GX41 antibody and analyzed by MALDI-TOF MS. K* refers to a lysine with a Gly-Gly modification. As shown above, the unmodified peptide is absent from the immunoprecipitate, while the peptide containing the Gly-Gly modification is immunoprecipitated.
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