Analytical gel filtration assays with SMC and ScpA and ScpB. A) Reference runs for purified SMC (0.3 nmol), ScpA (0.4 nmol) and ScpB (1 nmol), all three profiles were merged. Dashed lines show the position of peaks for proteins of gel filtration standards. The void volume peak is indicated by a white triangle. B-D) Coomassie stained SDS PAGE showing continuous fractions collected during gel filtration. Full or dashed lines show the regions from which the fractions were taken. E) ScpA mixed with ScpB in two different proportions, 1:2.5 nmol (lower graph) and 1:0.83 nmol (upper graph). Arrows indicate the existence of at least two discrete peaks containing different forms of the ScpA/B complex. F-G) Coomassie blue-stained SDS-PAGE showing continuous fractions collected during gel filtration. Dashed lines indicate the region from which the fractions were taken, full lines indicate the position of the fractions within the elution profile, indicating peak elution of ScpA, ScpB or of the distinct ScpA/B complexes. G) corresponds to (F) with a 1:10 ration of ScpA to ScpB. H) The elution peak at 12 ml is subjected to a second round of gel filtration analysis, and fractions containing ScpA and ScpB are shown (stained with silver), I) SMC (0.3 nmol) incubated with ScpA (0.4 nmol) and ScpB (1 nmol) before gel filtration. The dashed arrow shows the position of the SMC peak in the absence of Scps, and the solid arrow peak elution of the SMC complex. J) Silver-stained SDS-PAGE with (non-continuous) fractions loaded corresponding to elution peaks from gel filtration (indicated by solid lines) shown in I). Note that the first two lanes in (J) correspond to the first two lanes in (B) and in (F), i.e. that SMC shifts to a higher molecular weight in complex with ScpA/B, and the ScpA/B are not present in these fractions in the absence of SMC (F).
Mascarenhas et al. BMC Cell Biology 2005 6:28 doi:10.1186/1471-2121-6-28