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Polyubiquitin (K63-linkage-specific) monoclonal antibody (HWA4C4)

Key antibody for the detection of K63-linked polyubiquitinylated proteins.
BML-PW0600-0025 25 µl 109.00 USD
BML-PW0600-0100 100 µl 352.00 USD
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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

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
Figure 1: Western blot of single isopeptide-linkage polyubiquitinylated conjugates K6 (BML-UW0615), K11 (BML-UW0620), K27 (BML-UW0625), K29 (BML-UW0630), K33 (BML-UW0635), K48 (BML-UW0640), K63 (BML-UW0645),1µg each, using MAb to Polyubiquitinylated Conjugates (FK1) (BML-PW8805) and MAb to Polyubiquitin (K63-linkage-specific) (HWA4C4) (BML-PW0600) at 1:1’000 dilution.
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Figure 2: Western blot of HeLa S100 Fraction (BML-SW8750, 5µg) probed with MAb to Polyubiquitin (K63-linkage-specific) (HWA4C4) (BML-PW0600) (Lane A) and MAb to Polyubiquitinylated Conjugates (FK1) (BML-PW8805) (Lane B) at 1:1’000 dilution.
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Figure 3: Formalin-fixed paraffin-embedded sections of hippocampus from human Alzheimer’s disease brain stained with MAb to Polyubiquitin (K63-linkage-specific) (HWA4C4) at 1:100 dilution with biotinylated secondary antibody and DAB detection.
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Product Literature References

CYLD and the NEMO zinc finger regulate tumor necrosis factor signaling and early embryogenesis: Y. Zhao, et al.; J. Biol. Chem. 290, 22076 (2015), Application(s): Western blot , Abstract; Full Text
Cytosolic PTEN-INDUCED PUTATIVE KINASE 1 is Stabilized By NF-κB Pathway and Promotes Non-Selective Mitophagy: G.G. Lim, et al.; J. Biol. Chem. 290, 16882 (2015), Abstract; Full Text
Pale body-like inclusion formation and neurodegeneration following depletion of 26S proteasomes in mouse brain neurones are independent of α-synuclein: S.M. Paine, et al.; PLoS One 8, e54711 (2013), Application(s): Immunohistochemistry of mouse tissue, Abstract; Full Text

General Literature References

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin: H. Wang et al.; PNAS 105, 20197 (2008), Abstract; Full Text
Proteasome-independent functions of ubiquitin in endocytosis and signaling. : D. Mukhopadhyay & H. Riezman; Science 315, 201 (2007), Abstract;
Distinct monoubiquitin signals in receptor endocytosis. : K. Haglund et al. ; Trends Biochem.Sci. 28, 598 (2003), Abstract;
TAK1 is a ubiquitin-dependent kinase of MKK and IKK: C. Wang et al. ; Nature 412, 346 (2001), Abstract;
Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain. : J. Spence et al.; Cell 102, 67 (2000), Abstract;
Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. : R. M. Hofmann et al.; Cell 96, 645 (1999), Abstract;
A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. : J. Spence et al.; Mol.Cell Biol. 15, 1265 (1995), Abstract; Full Text

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