Application

Research CategorySignaling

Detect Ubiquitin using this Anti-Ubiquitin (pan) Antibody, clone 2G7B8 validated for use in Western Blotting.

Research Sub CategoryUbiquitin & Ubiquitin Metabolism

Western Blotting Analysis: 1.0 µg/mL from a representative lot detected the accummulation of ubiquitinated proteins in HEp-2 human epithelial cells following proteasome inhibitor MG-132 (Cat. No. 474790) treatment (Courtesy of Dr. Blagoy Blagoev, University of Southern Denmark).

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

General description

Ubiqutin (Ub) is initially produced as a 229-a.a. Polyubiquitin-B (UniProt P0CG47) precursor protein encoded by the UBB gene (Gene ID 7314) or a 685-a.a. Polyubiquitin-C precursor protein (UniProt P0CG48) encoded by the UBC gene (Gene ID 7316) in human. Posttranslational cleavages yield multiple copies of idential 76-amino acid ubiquitin (also known as high mobility group nonhistone protein HMG-20) molecules. Ub is involved in one of the most common post-translational modifications (PTMs) of cellular proteins, where Ub is linked covalently via its carboxyl terminus (Gly76) to usually Lys residues in target proteins. A given target Lys residue can be linked to one single Ub molecule (monoubiquitylated) or to a chain of Ub molecules (polyubiquitylated). In a polyUb chain, Ub molecules can be linked through one of the seven Lys residues (K6, K11, K27, K29, K33, K48, and K63) or through the Ub N-terminus Met1 residue (which generates linear chains). Lys-C is an endopeptidase that cleaves proteins on the C-terminal side of unmodified lysine residues, but not Ub-modified Lysine residues, generating peptide fragments with a C-terminus Lysine and internal Lysine residue(s) linked to a Ub C-terminal remnant (as a result of Lys-C cleavage between Ub K63 and E64). Antiboides that specifically target this Ub C-terminal remnant (i.e. E64 to Gly76 or E48 to Gly76 if K63 is ubiquitinated in a polyUb chain) are compatible with the patented UbiSite™ methodology for Lysine ubiquitination site determination developed by the laboratory of Dr. Blagoy Blagoev at University of Southern Denmark. Such antibodies can isolate Lys-C digested fragments with Ub modification site(s), followed by further trypsin digestion to leave only Gly75-Gly76 covalently linked to the modified Lysine on the isolated peptide fragments prior to Mass spectroscopy (MS) analysis to determine the Lysine modification site(s).

Immunogen

KLH-conjugated linear peptide corresponding to human ubiquitin C-terminal sequence.

Epitope: Near C-terminus.

Other Notes

Concentration: Please refer to lot specific datasheet.

Physical form

Purified mouse monoclonal IgG1κ antibody in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide.

Protein G Purified

Format: Purified

Quality

Evaluated by Western Blotting in HEp-2 human epithelial cell lysate.

Western Blotting Analysis: 2.0 µg/mL of this antibody detected ubiquitinated proteins in 10 µg of HEp-2 human epithelial cell lysate.

Specificity

Broad species reactivity expected based on 100% sequence homology.

Clone 2G7B8 detects monoubiquitinated (MonoUb) and polyubiquitinated (polyUb) proteins, as well as free ubiquitin (Ub) molecule by targeting Ub sequence C-terminal to Lys63 (UbiSite™ tag). Clone 2G7B8 can be used to detect (e.g. by Dot blot) and isolate (i.e. by immunoprecipitation) peptides derived from Ub-modified proteins after endoproteinase Lys-C digestion. Lys-C cleaves Ub and Ub-modified proteins after unmodified Lysine residues, but not Ub-modified Lysine residues, generating peptide fragments with their modified lysine residues linked to Ub C-terminal remnant (UbiSite™ tag) that cannot be recognized by other ubiquitin antibodies whose epitopes lie N-terminally to Lys63. The isolated peptide fragments can then be subjected to Mass spectroscopy (MS) analysis to allow identification of ubiquitin-modification sites.

Storage and Stability

Stable for 1 year at 2-8°C from date of receipt.

Target description

Variable depending on the size of the ubiquitinated protein(s)/peptide(s) and the degrees of ubiquitination.