Fig. 4.

Crosslinking between positions in the middle and C-terminal domains of FliG. (a) Examples of the crosslinking of double-Cys mutant proteins, using either plasmid-based expression (derivatives of pKP619 induced with 40 μM IPTG) or expression from the normal chromosomal locus. (b) Left: ladder of products observed with the chromosomally expressed 158/214 pair, resolved on a gel using less than the usual amount of bis-acrylamide (acrylamide:bis-acrylamide ratio 80:1). Right: Yield versus product size for the ladders produced by the 158/214 Cys pair. Bands were quantified by densitometry using Image-J [53]. (c) Blocking of the 158–214 crosslinking by pretreatment with NEM. Blocking was for 3 min prior to treatment with iodine for all except for the rightmost sample, which was blocked for 30 min. Proteins were resolved on a 4–20% gradient gel.