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Mono- and polyubiquitinylated conjugates monoclonal antibody (FK2) (HRP conjugate)

BML-PW0150-0025 25 µg 115.00 USD
BML-PW0150-0100 100 µg 385.00 USD
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  • Detects K29-, K48-, and K63-linked mono- and polyubiquitinylated proteins
  • Most cited multi-ubiquitin antibody with over 500 citations
  • Validated for WB, IP, IHC, and ELISA applications
FK2 antibody was conjugated to horseradish peroxidase (HRP) by a proprietary method. The HRP-linked FK2 monoclonal antibody (FK2H™) gives enhanced Western blotting detection of mono- and poly-ubiquitinylated protein conjugates and multi-ubiquitin chains compared to the unconjugated FK2 antibody (Prod. No. BML-PW8810). Shows no reactivity with free ubiquitin. Recognizes K29-, K48-, and K63-linked polyubiquitinylated and monoubiquitinylated proteins but not free ubiquitin.

By using the antibodies in concert the degree of protein ubiquitinylation may be determined. These antibodies have been used to not only investigate the dynamics of ubiquitin conjugation but also for the development of immunoassays permitting quantification of serum and intracellular multiubiquitin chains. The hybridoma secreting the antibody FK2 was generated by fusion of splenocytes from Balb/c mice which had received repeated immunization with a crude preparation of polyubiquitinylated-lysozyme. The antibody (clone FK2) has been extensively characterized by one-dimensional Western blotting. The immunoglobulin subclass is IgG1.

Product Specification

Immunogen:Crude preparation of polyubiquitinylated-lysozyme.
UniProt ID:P0CG47 (UBB), P0CG48 (UBC)
Species reactivity:Species independent
Specificity:Recognizes mono- and polyubiquitinylated protein conjugates in a wide range of species.
Crossreactivity:Does not cross-react with free ubiquitin.
Applications:ELISA, IHC, WB
Recommended Dilutions/Conditions:Western Blot (1:1,000)
Suggested dilutions/conditions may not be available for all applications.
Optimal conditions must be determined individually for each application.
Formulation:Liquid. In PBS.
Use/Stability:Antibody is supplied at a concentration of 1mg/mL. Store at 4ºC. Can be stored at -20ºC if required, but avoid multiple freeze/thawing. Vial contains purified HRP-linked immunoglobulin suspended in phosphate-buffered saline.
Handling:Avoid freeze/thaw cycles.
Shipping:Blue Ice Not Frozen
Long Term Storage:+4°C
Technical Info/Product Notes:FK2 antibody was conjugated to horseradish peroxidase (HRP) by a proprietary method. The HRP-linked FK2 monoclonal antibody (FK2H) gives enhanced western blotting detection of mono- and poly-ubiquitinylated protein conjugates and multi-ubiquitin chains compared to the unconjugated FK2 antibody (PW8810). For western blotting an initial antibody dilution of 1:1000 is recommended, followed by washing and signal detection using appropriate development reagents.
Note: Milk should NOT be used in blocking/antibody binding solutions. 1% BSA in PBS or TBS Tween should be used instead.
Figure: Lanes a-d, HeLa S3 S100 cytosol preparation (SW8750; 10µg per lane); lanes e-h, K48-linked multiubiquitin chains (Prod. No: BML-PW8860; 0.5µg per lane). Lanes a & e probed with Prod. No BML-PW8805 (clone FK1; 1µg/mL); lanes b & f probed with Prod. No. BML-PW0145 (FK1H™; 1µg/mL); lanes c & g probed with Prod. No. BML-PW8810 (clone FK2; 1µg/mL); lanes d & h probed with Prod. No. BML-PW0150 (FK2H™; 1µg/mL).
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Product Literature References

TSC2 N-terminal lysine acetylation status affects to its stability modulating mTORC1 signaling and autophagy: A. García-Aguilar , et al.; Biochem. Biophys. Acta 1863, 2658 (2016), Application(s): Western blot, Abstract;
Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages: M. Min, et al.; Mol. Biol. Cell 26, 4325 (2015), Application(s): Western Blot, Abstract;
PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis: M.S. Lee, et al.; Nat. Commun. 6, 7769 (2015), Application(s): Western Blot, Abstract; Full Text
Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme: A. Sarkar & I. Zohn; J. Cell Biol. 196, 789 (2012), Application(s): IF, WB of mouse embryonic tissue, Abstract; Full Text
Selective degradation of aggregate-prone CryAB mutants by HSPB1 is mediated by ubiquitin-proteasome pathways: H. Zhang, et al.; J. Mol. Cell. Cardiol. 49, 918 (2010), Application(s): WB of embryonic rat cardiac cells, Abstract; Full Text

General Literature References

Dynamics of ubiquitin conjugation during heat-shock response revealed by using a monoclonal antibody specific to multi-ubiquitin chains: M. Fujimuro et al.; Eur. J. Biochem. 249, 427 (1997), Abstract;
Serum concentrations of free ubiquitin and multiubiquitin chains: K. Takada et al.; Clin. Chem. 43, 1188 (1997), Abstract;
Immunoassay for the quantification of intracellular multi-ubiquitin chains: K. Takada et al.; Eur. J. Biochem. 233, 42 (1995), Abstract;
Production and characterization of monoclonal antibodies specific to multi-ubiquitin chains of polyubiquitinated proteins: M. Fujimuro et al.; FEBS Lett. 349, 173 (1994), Abstract;

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