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Additional resources for Applications of Chimeric Genes and Hybrid Proteins
Coli ALKALINEPHOSPHATASEGENE FUSIONS 35 expression of the downstream gene lacY. This permits an enrichment of interesting nonpolar, Lac-down mutations with specific effects on the fusion protein from the much larger class of nonsense mutations that simply abolish expression of both lacZ and lacY. 3° Conclusion Despite the usefulness of new reporter systems such as GFP, and the many epitope-tagging systems, the Lac system remains a powerful genetic tool both in studying gene regulation and in generating mutants in diverse complex biological systems.
Fluorescent Antibody Labeling for Flow Cytometry Strains and Reagents. , FACScan or FACScalibur); FACS analysis tubes: 12 x 75-mm round-bottom tubes. Phosphate-buffered saline (PBS); antibody (Ab)-staining buffer [5% bovine serum albumin (BSA) in PBS]; midlog cultures of bacteria; Ab 20 R. H. Valdivia and S. Falkow, Curr. Opin. Microbiol. 1, 359 (1998). ~ . ~, e ~ ~ ~ • . ~ ~ . -o~ . ~ . o~ ~ O o~=~ ~ . ~ ,- ~ ~ ~ . ~ . ~ . ~ ~ ~ ' ~ : ~ . ~ . ~ ~ ~ ~ ~ ~ o ~ . ~ ;~,~m m ~ ~ ~ ~ •'- o • ~ ~ 4= ~ ,_~ ;...
1 mM ZnC12, and 1 mM iodoacetamide in 13 × 100-mm glass tubes. Include a blank without cells. 1% sodium dodecyl sulfate and 50 /~1 chloroform, vortex, and incubate at 37 ° for 5 min to permeabilize cells. Place tubes on ice for 5 min to cool. 0) to each individual tube, agitate, and place in 37 ° water bath. Note time. 0, 1 M KH2PO4, and place the tube in an ice-water bath to stop the reaction. Note time. Measure OD550 and OD420for each sample, using an assay mixture without cells as a blank. 75 x OD55o)] x 1000 Units activity = time (min) × OD6oo X volume cells (ml) Acknowledgments We thank J.