By Simon M.I., Thorner J., Emr S.D.
The seriously acclaimed laboratory typical for greater than 40 years, equipment in Enzymology is among the such a lot hugely revered courses within the box of biochemistry. because 1955, each one quantity has been eagerly awaited, often consulted, and praised through researchers and reviewers alike. Now with greater than three hundred volumes (all of them nonetheless in print), the sequence includes a lot fabric nonetheless suitable this day - actually a necessary book for researchers in all fields of lifestyles sciences.
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Extra resources for Applications of Chimeric Genes and Hybrid Proteins: Cell Biology and Physiology
The choice of extraction procedure can, however, be readily assessed by preliminary comparisons using immunofluorescence. The major drawback with this extraction approach is that the plasma membrane is only poorly preserved. Also, on intracellular boundaries with membrane-associated complexes such as clathrin lattices, these complexes can prevent the penetration of probes such as antibodies to the membrane proteins that lie beneath them. Horseradish Peroxidase-Diaminobemidine Reaction on Living Cells To preserve HRP-containing compartments before subsequent extraction with digitonin or Triton X-100, the same concentrations of DAB and hydrogen peroxide are used as described above for cytochemistry, but the buffer is modified as follows and the reaction is carried out at 4”.
A. Surani, B. H. Hirst, and H. J. Gilbert, J. Biol. Chem. 265, lb996 i1990). HRP 131 CHIMERAS AS EM TRACERS 39 The HRP and c-myc KDEL fragments are ligated together and the product separated by electrophoresis on low gelling temperature (LGT) agarose. This product is then ligated into PSR WSS. Sequences of the human transferrin receptor, which include only the transmembrane domain and the cytoplasmic domain or only the transmembrane domain plus residues on either side of the signal anchor (residues 60-91) are generated by PCR from cDNA provided by I.
First, quantitation of intracellular distribution of an enzyme activity is both simpler and much more accurate than analysis by Western blotting or immunoprecipitation of gradient fractions. Moreover, the ease of handling large numbers of samples allows for rapid characterization of many different constructs, which has been important in determining the sequence dependence of various trafficking steps. 2-4 Second, data obtained by enzymatic assay are reproducible between independent experiments.
Applications of Chimeric Genes and Hybrid Proteins: Cell Biology and Physiology by Simon M.I., Thorner J., Emr S.D.