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 |
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Host: | Mouse |
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Isotype: | IgG2a |
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Immunogen: | Synthetic peptide containing the ubiquitin K63-isopeptide motif. |
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UniProt ID: | P0CG47 (UBB), P0CG48 (UBC) |
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Source: | Purified from hybridoma tissue culture supernatant. |
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Species reactivity: | Species independent
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Specificity: | Recognizes K63-linked polyubiquitin in wide range of species. |
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Crossreactivity: | Does not cross-react with any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyubiquitinylated species. |
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Applications: | ELISA, ICC, IHC, WB
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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. |
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Formulation: | Liquid. In PBS containing 0.09% sodium azide. |
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Handling: | Avoid freeze/thaw cycles. After opening, prepare aliquots and store at -20°C. |
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Shipping: | Blue Ice |
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Long Term Storage: | -20°C |
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Regulatory Status: | RUO - Research Use Only |
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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.
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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