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Polyubiquitinylated conjugates monoclonal antibody (FK1)

Widely used antibody for detection of polyubiquitinylated proteins.
BML-PW8805-0500 500 µg 389.00 USD
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The hybridoma secreting MAb to Polyubiquitinylated Conjugates (FK1) (Prod. No. BML-PW8805) was generated by fusion of splenocytes from Balb/c mice which had received repeated immunisation with a crude preparation of polyubiquitinylated-lysozyme. MAb to Polyubiquitinylated Conjugates (FK1) has been extensively characterised by one-dimensional Western blotting and has been shown to recognise only polyubiquitinylated proteins and not monoubiquitinylated proteins or free ubiquitin.

Product Specification

Immunogen:Poly-ubiquitinylated lysozyme.
UniProt ID:P0CG47 (UBB), P0CG48 (UBC), P62979 (RPS27A), P62987 (UBA52)
Species reactivity:Species independent
Specificity:Recognizes polyubiquitinylated conjugates.
Crossreactivity:Does not cross-react with mono-ubiquitinylated proteins or 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.
Purity Detail:Purified.
Formulation:Liquid. In PBS containing 0.1% sodium azide.
Use/Stability:PLEASE NOTE that this antibody is an IgM and it is important to ensure that a secondary antibody of appropriate specificity is used.
Handling:Store at 4°C when not in use. Use diluted antibody within 1 month. Do not freeze.
Shipping:Blue Ice Not Frozen
Long Term Storage:+4°C
Technical Info/Product Notes:In order to investigate the status and extent of protein ubiquitinylation it is necessary to have available a range of antibodies capable of differentiating firstly between free ubiquitin and ubiquitin-protein conjugates and, secondly and more importantly, between those that are mono-ubiquitinylated and those that are poly-ubiquitinylated. The antibodies PW8805 (clone FK1) and PW8810 (clone FK2), originally developed in the laboratory of Professor Hideyoshi Yokosawa at Hokkaido University, Japan, are specific for ubiquitin-protein conjugates and show no reactivity with free ubiquitin. The hybridoma secreting the antibody FK1 was generated by fusion of splenocytes from Balb/c mice which had received repeated immunisation with a crude preparation of polyubiquitinylated-lysozyme. The antibody (clone FK1) has been extensively characterised by one-dimensional Western blotting (see Figure) and has been shown to recognise only polyubiquitinylated proteins and not monoubiquitinylated proteins or free ubiquitin. The immunoglobulin subclass is IgM. Clone FK1 recognises only polyubiquitinylated proteins and not monoubiquitinylated proteins or free ubiquitin, whilst clone FK2 recognises both mono- and poly-ubiquitinylated species but not free ubiquitin, thus by using the antibodies in concert the degree of protein ubiquitinylation may be determined.

ELISA: 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. Immunoblotting: For Western blotting an initial dilution of 1:1000 is recommended. Note: Milk should NOT be used in blocking/antibody binding solutions. 1% BSA in PBS or TBS Tween should be used instead.
Figure 1: Western blot of multi-ubiquitin chains using MAb to Polyubiquitinylated Conjugates (FK1) (Prod. No. BML-PW8805) (lanes A-C) and MAb to Mono- and Polyubiquitinylated Conjugates (FK2) (Prod. No. BML-PW8810) (lanes D-F).

Lanes A & D: K48-linked chains.Lanes B & E: K29-linked chains. Lanes C & F: K63-linked chains.

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Product Literature References

Effectiveness of daily eccentric contractions induced via kilohertz frequency transcutaneous electrical stimulation on muscle atrophy: M. Tanaka, et al.; Acta Histochem. 118, 56 (2016), Application(s): Western Blot, Abstract;
Zyxin-Siah2–Lats2 axis mediates cooperation between Hippo and TGF-β signalling pathways: B. Ma, et al.; Nat. Commun. 7, 11123 (2016), Application(s): Immunoblotting, Abstract;
A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis: X. Tian, et al.; PLoS One 13, e1002103 (2015), Application(s): Immunohistochemistry , Abstract; Full Text
Complete and ubiquitinated proteome of the Legionella-containing vacuole within human macrophages: W.M. Bruckert, et al.; J. Proteome. Res. 14, 236 (2015), Application(s): Labeled LCVs, Abstract; Full Text
Particulate cytoplasmic structures with high concentration of ubiquitin-proteasome accumulate in myeloid neoplasms: A. Pecci, et al.; J. Hematol. Oncol. 8, 71 (2015), Application(s): Immunogold Analysis, Abstract; Full Text
Phosphorylation of the deubiquitinase USP20 by Protein Kinase A regulates post-endocytic trafficking of β 2 adrenergic receptors to autophagosomes during physiological stress: R.P. Kommaddi, et al.; J. Biol. Chem. 290, 8888 (2015), Application(s): Western Blotting, Abstract; Full Text
Sestrin 2 Regulates PDGF Receptor beta (PDGFRβ) Expression by Modulating Proteasomal and Nrf2 Transcription Factor Functions: A. Tomasovic, et al.; J. Biol. Chem. 290, 9738 (2015), Abstract; Full Text
Structure of the Legionella Virulence Factor, SidC Reveals a Unique PI(4)P-Specific Binding Domain Essential for Its Targeting to the Bacterial Phagosome: X. Luo, et al.; PLoS Pathog. 11, e1004965 (2015), Application(s): Western Blot, Abstract; Full Text
Activation of the cAMP/PKA pathway induces UT-A1 urea transporter monoubiquitination and targets it for lysosomal degradation: H. Su, et al.; Am. J. Physiol. Renal Physiol. 305, F1775 (2013), Application(s): WB of rat kidney inner medullary tissue, Abstract;
Mitochondrial quality, dynamics and functional capacity in Parkinson's disease cybrid cell lines selected for Lewy body expression: G. Cronin-Furman, et al.; Mol. Neurodegener. 8, 6 (2013), Application(s): IHC of human neural cell model , Abstract; Full Text
Role of TAp73 in Female Reproductive Aging and Fertility: T. Yavorska; (2013), (Thesis paper), Application(s): WB of TAp73 in mouse oocytes, Abstract;
Ubiquitin-dependent recruitment of the Bloom syndrome helicase upon replication stress is required to suppress homologous recombination: S. Tikoo, et al.; EMBO J. 32, 1778 (2013), Application(s): WB of human cells from BS patients, Abstract; Full Text
Hectd1 regulates intracellular localization and secretion of Hsp90 to control cellular behavior of the cranial mesenchyme: A. Sarkar, et al.; J. Cell Biol. 196, 789 (2012), Application(s): WB of rabbit serum, Abstract; Full Text
NF-κB activation and polyubiquitin conjugation are required for pulmonary inflammation-induced diaphragm atrophy: A. Haegens, et al.; Am. J. Physiol. Lung Cell. Mol. Physiol. 302, L103 (2012), Application(s): WB, PCR of mouse diaphragm, Abstract; Full Text
53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress: C. Lukas, et al.; Nat. Cell Biol. 13, 243 (2011), Application(s): WB, PCR, Abstract;
Fibroblast growth factor receptor 3 (FGFR3) is a strong heat shock protein 90 (Hsp90) client: implications for therapeutic manipulation: M. Laederich, et al.; J. Biol. Chem. 286, 19597 (2011), Application(s): IP, IF of plasmids containing mouse and human growth factors, Abstract; Full Text
Histone crosstalk directed by H2B ubiquitination is required for chromatin boundary integrity: M. Ma, et al.; PLoS Genet. 7, e1002175 (2011), Application(s): WB, IP, PCR of chicken chromatin region , Abstract; Full Text
p23H implicated as cis/trans regulator of AlaXp-directed editing for mammalian cell homeostasis: M. Nawaz, et al.; Proc. Natl. Acad. Sci. USA 107, 2723 (2011), Application(s): WB, PCR, and Cell Culture of mouse tissue, Abstract; Full Text
p62 and NDP52 Proteins Target Intracytosolic Shigella and Listeria to Different Autophagy Pathways: S. Mostowy, et al.; J. Biol. Chem. 286, 26987 (2011), Application(s): WB, FLOW, IP in bacteria, Abstract; Full Text
PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1: S. Geisler, et al.; Nat. Cell Biol. 12, 119 (2010), Abstract;
Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation: K. Haglund et al.; Nat. Cell. Biol. 5, 461 (2003), Abstract;
The FMR1 CGG repeat mouse displays ubiquitin-positive intranuclear neuronal inclusions; implications for the cerebellar tremor/ataxia syndrome: R. Willemsen et al.; Hum. Mol. Genet. 12, 949 (2003), Abstract;
Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein-ubiquitin conjugates: N. Bishop et al.; J. Cell. Biol. 157, 91 (2002), Abstract;
PI31 is a modulator of proteasome formation and antigen processing: D. M. W. Zaiss; PNAS 99, 14344 (2002), Abstract;
Transient aggregation of ubiquitinated proteins during dendritic cell maturation: H. Lelouard et al.; Nature 417, 177 (2002), Abstract;
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; Full Text
Serum concentrations of free ubiquitin and multiubiquitin chains: K. Takada et al.; Clin. Chem. 43, 1188 (1997), Abstract; Full Text
Immunoassay for the quantification of intracellular multiubiquitin chains: K. Takada et al.; Eur. J. Biochem. 233, 42 (1995), Abstract; Full Text
Production and characterisation of monoclonal antibodies specific to multiubiquitin chains of polyubiquitinated proteins. : M. Fujimuro et al.; FEBS Letts. 349, 173 (1994), Abstract;

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