FIG. 4.

Affinity blotting of cell-derived proteins, performed with representative recombinant cohesins from ScaA, ScaB, and ScaC as the probes. A. cellulolyticus cells were grown on cellobiose and separated by centrifugation into supernatant (Sup) and pellet (Cell) fractions. The supernatant fraction was further fractionated by adsorption onto amorphous (phosphoric acid-treated) cellulose prior to subsequent electrophoresis. Samples (cell-associated pellet or cellulose-adsorbed supernatant) were subjected to SDS-PAGE (Gel) and were blotted onto nitrocellulose membranes (Blots). Gels were stained with Coomassie brilliant blue. The blots were probed with different recombinant protein samples, and labeled bands were detected by chemiluminescence by using peroxidase-conjugated, anti-His tag antibody. The probes were CohA5 (the fifth cohesin of ScaA), CohB1 (the first cohesin of ScaB), and CohC3 (the third cohesin of ScaC). Lane Std contained prestained protein molecular weight markers.