| 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 proteins 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. Applications: Suitable for use in ELISA and Western Blot. Other applications not tested. Recommended Dilutions: ELISA: 1:1000-1:5000 Western Blot: 1:200-1:1000 Optimal dilutions to be determined by the researcher. Storage and Stability: Lyophilized powder may be stored at -20C. Stable for 12 months at -20C. Reconstitute with sterile ddH2O. Aliquot to avoid repeated freezing and thawing. Store at -20C. Reconstituted product is stable for 12 months at -20C. 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. |
