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. 2012 Mar 12;109(13):4857-4862. doi: 10.1073/pnas.1118157109

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Fig. 4. — In vitro oligomerization assay. (A) A representative cross-linking experiment. 11 μM of WT DHFR, W133V, or I155A purified proteins in 25 mM potassium-phosphate buffer (pH 7.8) and 100 μM NADPH were incubated for 45 min at room temperature (RT) or 42 °C with or without 2 mM of cross-linking (c-l) agent (glutaraldehyde). Shown is the SDS/PAGE analysis of 10 μL of protein samples after Coomassie staining. Red arrows indicate molecular weight equivalent of monomeric and oligomeric protein species. Smears at high molecular weight seen for W133V protein at 37 °C and 42 °C in the presence of c-l are due to extensive aggregation. (B) The in vitro propensity to oligomerize is correlated to fitness values measured by competition at 42 °C. Oligomerization propensity was determined by measuring the relative density of the SDS/PAGE bands corresponding to all DHFR oligomerized species for each of the purified mutant (Fig. S8). The degree of oligomerization for every mutant is normalized to WT DHFR. (C) Correlation of oligomerization propensity (as measured in B) to mutant’s .

Inline graphic — In vitro oligomerization assay. (A) A representative cross-linking experiment. 11 μM of WT DHFR, W133V, or I155A purified proteins in 25 mM potassium-phosphate buffer (pH 7.8) and 100 μM NADPH were incubated for 45 min at room temperature (RT) or 42 °C with or without 2 mM of cross-linking (c-l) agent (glutaraldehyde). Shown is the SDS/PAGE analysis of 10 μL of protein samples after Coomassie staining. Red arrows indicate molecular weight equivalent of monomeric and oligomeric protein species. Smears at high molecular weight seen for W133V protein at 37 °C and 42 °C in the presence of c-l are due to extensive aggregation. (B) The in vitro propensity to oligomerize is correlated to fitness values measured by competition at 42 °C. Oligomerization propensity was determined by measuring the relative density of the SDS/PAGE bands corresponding to all DHFR oligomerized species for each of the purified mutant (Fig. S8). The degree of oligomerization for every mutant is normalized to WT DHFR. (C) Correlation of oligomerization propensity (as measured in B) to mutant’s .