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. Author manuscript; available in PMC: 2013 Sep 13.
Published in final edited form as: J Mol Biol. 2012 Mar 3;419(0):89–101. doi: 10.1016/j.jmb.2012.02.043

Figure 4.

Figure 4

Oligomerization state of wild type and mutant OmpF's. OmpF proteins were expressed, purified, and refolded from inclusion bodies as described in Methods. Folding reactions were quenched by adding 5× SDS gel-loading buffer [45] to a final dilution of 1× SDS gel loading buffer. 50 μl of sample was loaded on a 4-20% acrylamide continous gradient precast gel to resolve the folded and unfolded populations [12]. a) The wild type and mutants (G57A/G59A and G57S/G59S) were only present in either folded trimeric state (T) or unfolded state (U). The mutants R100L/G19W and G57I/G59L have folded monomeric (M) and unfolded species. The mutant R100L/G135W exists in folded dimeric (D), monomeric, as well as an unfolded state. b) After overnight refolding, subsequent degradation of the unfolded protein was induced by the addition of trypsin (trypsin/protein 1:100 w/w) [44]. Final protein was purified and concentrated using Centrifugal Filters. The folded oligomeric and monomeric bands were resistant to trypsin digestion, while the unfolded band disappeared after treatment with trypsin.