By Melvin I. Simon
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Extra resources for Applications of Chimeric Genes and Hybrid Proteins, Part B: Cell Biology and Physiology
Incubate for 1 hr at room temperature. 8. Wash the slides twice in PBS. 9. Apply a horseradish peroxidase–avidin conjugate, and incubate for 20 min. 10. Wash the slides twice in PBS. 11. Add the substrate solution, and incubate for 5 min. 12. Wash the slides. 13. Counterstain with hematoxylin, using standard techniques. 14. Wash the slide, apply Aquamount and a coverslip, and allow to dry. Considerations. If this is the ﬁrst foray of the reader into immunohistochemistry, enlistment of a collaborator in a dedicated histology laboratory would be well advised.
RIORDAN AND BERT L. VALLEE VOLUME 228. Aqueous Two-Phase Systems Edited by HARRY WALTER AND GO¨TE JOHANSSON VOLUME 229. Cumulative Subject Index Volumes 195–198, 200–227 VOLUME 230. Guide to Techniques in Glycobiology Edited by WILLIAM J. LENNARZ AND GERALD W. HART VOLUME 231. Hemoglobins (Part B: Biochemical and Analytical Methods) Edited by JOHANNES EVERSE, KIM D. VANDEGRIFF, AND ROBERT M. WINSLOW VOLUME 232. Hemoglobins (Part C: Biophysical Methods) Edited by JOHANNES EVERSE, KIM D. VANDEGRIFF, AND ROBERT M.
If the tag is to be placed at the N or C terminus of the protein, the researcher can employ one of many publicly or commercially available vectors. Many modern cloning vectors contain one or more epitope tags ﬂanking the polylinker, suitably coupled to bacterial and/or eukaryotic promoters and transcription terminators. Use of these sorts of vectors allows a single cloning step in which the coding sequence is ligated into the polylinker. DNA sequencing across the cloning junction and a quick Western analysis of extract from a transfected line are then used to conﬁrm the integrity of the resulting construct.
Applications of Chimeric Genes and Hybrid Proteins, Part B: Cell Biology and Physiology by Melvin I. Simon