By Simon M.I., Thorner J., Emr S.D.
The seriously acclaimed laboratory general for greater than 40 years, equipment in Enzymology is without doubt one of the such a lot hugely revered courses within the box of biochemistry. for the reason that 1955, each one quantity has been eagerly awaited, usually consulted, and praised via researchers and reviewers alike. Now with greater than three hundred volumes (all of them nonetheless in print), the sequence comprises a lot fabric nonetheless suitable this present day - really a vital ebook for researchers in all fields of lifestyles sciences.
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Additional info for Applications of Chimeric Genes and Hybrid Proteins: Cell Biology and Physiology
Hazlewood, M. 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.
9% (w/v) NaCl is used. 4 *‘A Gibson Dbmingo, C. E. Futter, S. Maxwell, E. H. Allchin, M. -P. Kraehenbuhl, D. ‘G. Odorizzi, I. S. Trowbridge, and C. R. Hopkins, J. Cell Biol. 143, 81 (1998). 2% (w/v) sodium azide for 10 min at 4”. Cells are then washed three times in PBS+ at 4” before fixation in PBS+ containing 2% (w/v) paraformaldehyde at 4” but are then allowed to warm to room temperature during a 15min incubation period in fixative. 2) and then incubated in permeabilization buffer containing digitonin (40 pg/ml; diluted from a lOOmg/ml stock in DMSO) and protease inhibitor cocktail for lo-20 min (depending on the cell type) at 4”.
Proceed to step 3. 2b. 1% (v/v) Triton X-100 for 15 min to permeabilize the cells. Proceed to step 3. 3. Add 4 ml of AP fusion protein and incubate at room temperature for 90 min. The time is determined by the rate of binding, k,,. For the reaction of a cell surface receptor and a polypeptide ligand, k,, can be quite slow, but 60 to 90 min at room temperature should be enough to give I4 S. Davis, J. Bruno, (1996). T. H. Aldrich, C. Radziejewski, P. F. Jones, A. Acheson, P. C. Maisonpierre, D. L.
Applications of Chimeric Genes and Hybrid Proteins: Cell Biology and Physiology by Simon M.I., Thorner J., Emr S.D.