FIG. 2.

Western blot analysis of small protein levels. (A) Proteins induced by SDS and/or EDTA exposure. Cells grown overnight in LB were diluted into 30 ml of LB and grown to an OD₆₀₀ of 0.2 to 0.3. Cultures were then split into five 5-ml aliquots and exposed to water, 0.025% SDS, 1 mM EDTA, or 0.025% SDS plus 1 mM EDTA. Cells were harvested before stress (T0) and at an OD₆₀₀ of 1.2 to 1.7. (B) Proteins induced under acidic conditions. Cells grown overnight in LB were diluted into 30 ml of either LB-MOPS (pH 7.5) or LB-MES (pH 5.5) and harvested at an OD₆₀₀ of 0.3 to 0.4. (C) Proteins induced by heat shock. Cells grown overnight in LB at 30°C were diluted into 30 ml of LB and grown at 30°C to an OD₆₀₀ of 0.4. Cultures were then split into three 10-ml aliquots. One set of samples was transferred to 45°C, while the other half was kept at 30°C. Cells were harvested before transfer (T0) as well as 5 and 20 min after transfer. Western blot analysis using anti-FLAG, alkaline phosphatase-conjugated antibodies was carried out with whole-cell extracts harvested from the cultures above. Asterisks denote the band corresponding to the fusion protein. Exposure times were optimized for each panel for visualization here; therefore, the signal intensity shown does not indicate relative abundance between proteins.