Figure 3. PEG-mal labeling of periplasmic and cytoplasmic ArnT amino acids.

(a, b, and d) E. coli DH5α carrying plasmids encoding ArnT cysteine replacements were grown to mid-exponential phase and protein expression induced with 0.2% l-arabinose. Cells were harvested and resuspended in 0.3 ml of HEPES/MgCl₂ buffer. 0.1 ml of cell suspension was incubated with buffer alone or 1 mM PEG-mal with or without 2% SDS for 1 h at room temperature. Reactions were quenched with 45 mM DTT, and proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes, followed by immunoblot with anti-FLAG antibodies. (c) Crude membrane fractions were incubated with buffer alone or pre-treated with 5 mM NEM, after that membrane were incubated with 1 mM PEG-mal and 2% SDS for 1 h at room temperature. Reactions were quenched with 45 mM DTT. (e) Crude membrane fractions were isolated by ultracentrifugation and divided in two aliquots. One aliquot was incubated with buffer alone and the other with 1 mM PEG-mal. The reactions were quenched with 45 mM DTT and separated by SDS-PAGE. Immunoblot was probed with anti-FLAG antibody.