By Ian A. McKay, Kenneth D. Brown
Development components and Receptors: a realistic technique offers finished protocols for reviews of progress components and their interactions with receptors. It covers a variety from easy analytical suggestions to classy in vivo functions together with: RT-PCR and immunocytochemistry for detection of development elements and receptors; construction and purification of recombinant progress components and receptors; labelling of progress elements for binding reports; in vivo mutagenesis; the yeast two-hybrid assay of protein-protein interactions; phage show of things; program of things to wound-healing approaches utilizing the gene gun; remedy of cancers with factor/toxin chimeras; and research of significant issue domain names utilizing chimeric proteins. This booklet updates and extends the present literature and describes very important novel methods to the examine of development components and their receptors, together with using RNA aptamers as receptor antagonists, and the advance of receptor superantagonists. will probably be of large price to researchers and lecturers and, with an appendix that lists a good number of development components and receptors, will function a convenient reference textual content.
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Additional resources for Growth Factors and Receptors: A Practical Approach (Practical Approach Series)
So far, there have been three Trk receptors characterized which interact with neurotrophins. TrkA, TrkB, and TrkC serve as receptors for NGF, BDNF, and NT3 respectively. In addition, TrkB is shared by NT-4 and BDNF. Another receptor with which all the neurotrophins to similar extents interact, is the low affinity receptor, called p75. The role of p75 in neurotrophin signalling is not understood (2, 3). However, there have been recent studies suggesting a signalling mechanism involving NF-kB activation and production of ceramide, but the details of these pathways remain obscure (4, 5).
Typical final yield is about 50-100 ug of pure protein per litre of conditioned medium. aThe gradient may be adjusted for optimal separation. ''Neurotrophins usually elute at around 40% acetonitrile (RP buffer B). 6. Biochemical assays Constructing and expressing viable proteins would be futile if we have no means by which to measure the effects of mutations on the function of the protein. We may also want to evaluate whether introduced mutations confer new activities to the parent molecule. Consequently, biochemical assays suitable for assessing protein funtion are essential.
Some groups have even tried modifying sequences around the translation initiation region to improve secretion of heterologous proteins (37). 5. Protein purification Obtaining pure protein is not always necessary in order to characterize some activities of the protein, or if the aim is to compare the behaviour of a mutant with wild-type protein. It is generally advisable, particularly for a long series of mutants, to assay the activity of proteins transiently produced in COS cell conditioned medium.
Growth Factors and Receptors: A Practical Approach (Practical Approach Series) by Ian A. McKay, Kenneth D. Brown