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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	143.00 USD

BML-PW0600-0100	100 µl	450.00 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 Details

Clone:	HWA4C4

Host:	Mouse

Isotype:	IgG2a

Immunogen:	Synthetic peptide containing the ubiquitin K63-isopeptide motif.

UniProt ID:	P0CG47 (UBB), P0CG48 (UBC)

Source:	Purified from hybridoma tissue culture supernatant.

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:	Blue Ice

Long Term Storage:	-20°C

Regulatory Status:	RUO - Research Use Only

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

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.

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.

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

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

Legionella longbeachae Regulates the Association of Polyubiquitinated Proteins on Bacterial Phagosome with Multiple Deubiquitinases: Y. Shi, et al.; Microbial. Spectr. 11, 04179 (2023), Abstract;

MLN4924 Inhibits Defective Ribosomal Product Antigen Presentation Independently of Direct NEDDylation of Protein Antigens: K. Vijayasimha, et al.; J. Immunol. 208, 2273 (2022), Abstract;

N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization: H. Shi, et al.; Nat. Commun. 12, 1379 (2021), Abstract;

BLM Potentiates c-Jun Degradation and Alters Its Function as an Oncogenic Transcription Factor: R. Priyadarshini, et al.; Cell Rep. 24, 947 (2018), Abstract;

Embryonic lethality in mice lacking Trim59 due to impaired gastrulation development: X. Su, et al.; Cell Death Dis. 9, 302 (2018), Abstract; Full Text

Salmonella escapes antigen presentation through K63 ubiquitination mediated endosomal proteolysis of MHC II via modulation of endosomal acidification in dendritic cells: M. Gogoi, et al.; Pathog. Dis. 76, ftx1258 (2018), Abstract; Full Text

The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome: F. Humphries, et al.; Nat. Commun. 9, 1560 (2018), Abstract; Full Text

ZUFSP Deubiquitylates K63-Linked Polyubiquitin Chains to Promote Genome Stability: P. Haahr, et al.; Mol. Cell 70, 165 (2018), Abstract;

A microchip platform for structural oncology applications: C.E. Winton, et al.; NPJ Breast Cancer 2, 16016 (2016), Abstract; Full Text

Formaldehyde Is a Potent Proteotoxic Stressor Causing Rapid Heat Shock Transcription Factor 1 Activation and Lys48-Linked Polyubiquitination of Proteins: S. Ortega-Atienza, et al.; Am. J. Pathol. 186, 2857 (2016), Abstract; Full Text

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

pVHL Mediates K63-Linked Ubiquitination of nCLU: J. Xue, et al.; PLoS One 7, e35848 (2012), Abstract; Full Text

K33-linked polyubiquitination of T cell receptor-zeta regulates proteolysis-independent T cell signaling: H. Huang, et al.; Immunity 33, 60 (2010), Application(s): Western blot, 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