A beginner's guide to Forth by David Johns

By David Johns

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And Weissman, A. M. (2004). A field guide to ubiquitylation. Cell Mol. Life Sci. 61, 1546–1561. Fischer, J. A. (2003). Deubiquitinating enzymes: Their roles in development, differentiation, and disease. Int. Rev. Cytol. 229, 43–72. Galloway, C. , Sowden, M. , and Smith, H. C. (2003). Increasing the yield of soluble recombinant protein expressed in E. coli by induction during late log phase. Biotechniques 34, 524–526, 528, 530. , Pan, Z. , and Wilkinson, K. D. (2003). Identification and characterization of DEN1, a deneddylase of the ULP family.

Once the chain has reached its final length, the distal C48 residue can be alkylated with ethyleneimine (above) or iodoacetamide, if desired, to reduce the potential for precipitation. 6). Excess iodoacetamide should be removed by repeated concentration/dilution or dialysis. Synthesis of K63‐Linked Chains The principles and procedures are similar to those outlined for K48‐ linked chains, with several differences. First, the synthetic reaction contains 8 M each of yeast Mms2 and Ubc13 in place of E2‐25K.

2000). This chapter also describes expression of His6Ub and purification of His6Ub protein conjugates from a eukaryotic cell, Saccharomyces cerevisiae, to identify ubiquitinated proteins by immunological methods. His6Ub, conjugated to proteins in vivo can also be used to identify previously unknown substrates of the ubiquitin pathway (see other issues in this volume for proteomic uses of tagged ubiquitin). Production of a His6Ub Open Reading Frame for Expression in E. coli or for Multimeric Expression in Eukaryotic Cells Although there are commercially available vectors with hexahistidine‐ encoding nucleotides followed by restriction endonuclease sites to facilitate cloning ORFs in‐frame, many contain additional nucleotides that add amino acids to the ORF.

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