BIOTECHNOLOGY
Fig. 3 shows how serine/threonine-
protein kinase AKT plays a role in many cellular processes. Survival factors can suppress apoptosis in a transcription- independent manner by activating the serine/threonine kinase AKT1, which then phosphorylates and inactivates components of the apoptotic machinery. (60203-2-Ig detects all the AKT members with or without phosphorylation, 66444- 1-Ig detects the phospho-Ser473 of AKT1 and phospho-S474 of AKT2/phospho- Ser472 of AKT3.) Ubiquitination. Ubiquitin is a small (+/-8.6 kDa) protein expressed across almost all tissue types, which covalently binds to lysine, cysteine, serine, threonine, or directly to the protein N-terminus through an enzymatic reaction catalysed by a three-enzyme cascade (E1, E2, and E3). Te enzyme cascade provides substrate specificity and activation, conjugation, and ligation steps. Proteins may be mono-ubiquitinated, or additional ubiquitin molecules may bind to the initial ubiquitin molecule, causing poly- ubiquitination. Ubiquitination can mark proteins for degradation and is also important for cellular signalling, the internalisation of membrane proteins and the development
Fig. 2
and regulation of transcription. Ubiquitin can be removed from proteins by deubiquitinating enzymes, which then lowers the molecular weight (Fig. 4). Fig. 4 demonstrates how ubiquitin B (UBB) (10201-2-AP), a member of the ubiquitin family, is required for ATP- dependent, non-lysosomal intracellular protein degradation of abnormal proteins
and normal proteins with a rapid turnover. Tis gene consists of three direct repeats of the ubiquitin coding sequence, with no spacer sequence.
Fig. 3 50
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PROTEIN COMPLEXES As Western blotting SDS-PAGE is performed in denaturing conditions, most protein complexes that are composed of proteins linked via non- covalent bonds disassociate during sample preparation and electrophoresis, with the component proteins then running as monomers. However, some proteins remain partially or fully present in homo- or hetero- meric complexes, even in the presence of SDS and β-Mercaptoethanol. In these cases the observed molecular weight can be substantially higher than the predicted, calculated monomeric form (Fig. 5). Some proteins, especially transmembrane proteins and proteins with hydrophobic domains, can aggregate during cell lysis as they are released from their native protein complexes and lipid membranes. Tese aggregates have high molecular weights and may not represent interactions that occur in their native states.
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