Ubiquitin

Ubiquitin (Ub) is a small, 76-residue, protein (8.5kD) found both as free monomer and covalently attached to itself and other proteins in eukaryotic cells. Free Ub is a very compact and stable molecule that is easily refolded after being denatured. It is therefore recommended that for detection of free Ub on Westerns, the Tris-Tricine SDS-PAGE is used and nitrocellulose filters are autoclaved after the transfer and before blocking and addition of anti-Ub antibodies. The C-terminus of ubiquitin forms an isopeptide bond with the e-amino group of a lysine side chain in a target protein. In this way proteins can be covalently modified by the addition of ubiquitin which may alter the target protein's function. Monoubiquitination generally targets proteins for internalization, endocytosis and lysosomal degradation, or modifies the surface charge of histones and affects chromatin compaction. If a chain of multiple copies of ubiquitin is attached to a protein, this appears to target the protein for degradation by the large intacellular protease known as the 26S proteasome. The process of intracellular proteasomal proteolysis is very rapid and reversible which makes detection of certain ubiquitinated proteins extremely difficult. Several specific inhibitors of proteasomes are commercially available and have been proven to be very useful in research applications. It is therefore recommended that cell lysates are kept on ice and the lysis buffers contain one or more of cysteine protease inhibitors.\n\nApplications: \nSuitable for use in ELISA and Western Blot. Other applications not tested.\n\nRecommended Dilutions: \nELISA: 1:1000-1:5000\nWestern Blot: 1:200-1:1000\nOptimal dilutions to be determined by the researcher.\n\nStorage and Stability:\nLyophilized powder may be stored at -20 degrees C. Stable for 12 months at -20 degrees C. Reconstitute with sterile ddH2O. Aliquot to avoid repeated freezing and thawing. Store at -20 degrees C. Reconstituted product is stable for 12 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

U0999

Size

500ug

Applications

ELISA, WB

Hosts

Rabbit

Form

Supplied as a lyophilized powder from PBS, pH 7.2, 0.01% sodium azide. Reconstitute with 100ul sterile ddH2O. No stabilizing proteins added.

P Type

Pab

Purity

Purified by delipidation, salt fractionation and ion exchange chromatography.

Isotype

IgG

References

1. Mimnaugh, E.G.,et al., (1999) Electrophoresis 20:418-428. 2. Wilkinson, K.D. (2000) Cell Dev. Bio. 11:141-148. 3. Wilkinson, K. D., and Hochstrasser, M. (1998) in Ubiquitin and the Biology of the Cell (Peters, J. M., Harris, J. R., and Finley, D., eds) , pp. 99-125, Plenum Press, New York. 4. Mimnaugh, E.G., Neckers, L.M. (2002) Methods Mol Biol. 194:179-203.

Additional Info

Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-rabbit serum.