Description:

Modification of proteins by addition of K⁶³-linked polyubiquitin chains is implicated in a variety of cellular events, including DNA repair, signal transduction and receptor endocytosis.

A monoclonal antibody (mAb) has been generated that specifically recognizes K63-linked polyubiquitin chains, but not any other isopeptide-linked (K⁶, K¹¹, K²⁷, K²⁹, K³³, or K⁴⁸) polyubiquitin chain or monoubiquitin. The specificity of this K⁶³-linked ubiquitinspecific monoclonal antibody was demonstrated using synthetic ubiquitin chains, single isopeptide-linked polyubiquitin chains, and with endogenously ubiquitinylated species in both cell lysates and intact cells.

The hybridoma secreting the antibody [clone HWA4C4] was generated by fusion of splenocytes from Balb/c mice which had received repeated immunisation with a synthetic peptide encompassing the ubiquitin K⁶³-isopeptide motif. Immunoglobulin was purified from tissue culture supernatant by ion exchange chromatography.

Product Specification:

Clone:	HWA4C4
Host:	Mouse
Isotype:	IgG2a
Immunogen:	Synthetic peptide containing the ubiquitin K63-isopeptide motif.
UniProt ID:	P0CG47 (UBB), P0CG48 (UBC)
Species reactivity:	Species independent
Specificity:	Recognizes K63-linked polyubiquitin in wide range of species.
Crossreactivity:	Does not cross-react with any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyubiquitinylated species.
Applications:	ELISA, ICC, IHC, WB
Recommended Dilutions/Conditions:	Immunocytochemistry (1:100) Immunohistochemistry (1:100) Western Blot (1:500 - 1:1,000) Specificity for K63 of mAb to Polyubiquitin (K63-linkage-specific) (HWA4C4) is temperature sensitive. Western blot membranes must not be autoclaved. Excessive heat should be avoided in all applications. K63-linked polyubiquitin modified proteins comprise only a proportion of total cellular polyubiquitinylated species. Increased sample loading may therefore be required for analysis by Western blot. Do not use milk as blocking solution. Use 1% BSA in an appropriate buffer instead. Suggested dilutions/conditions may not be available for all applications. Optimal conditions must be determined individually for each application.
Formulation:	Liquid. In PBS containing 0.09% sodium azide.
Handling:	Avoid freeze/thaw cycles. After opening, prepare aliquots and store at -20°C.
Shipping:	Shipped on Blue Ice
Long Term Storage:	-20°C

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