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. 1999 Jan 19;96(2):349–354. doi: 10.1073/pnas.96.2.349

Figure 2.

Figure 2

Purification of PYR complex from MEL nuclear extract. (A) Purification scheme. PYR complex was purified from 800 mg of MEL crude nuclear extract in five chromatographic steps, using continuous NaCl gradients (diagonal lines) to elute PYR complex activity from each column. (B) Analysis of fractions from the final two purification steps by gel shift assay (Upper) and silver-stained 7.5% SDS/PAGE (Lower). Fractions from the double-stranded DNA (dsDNA) cellulose column containing peak PYR complex activity (fractions 6–8, Left) were pooled and applied to a 1-ml δ60ym DNA-affinity column. High molecular weight PYR complex activity peaks off the affinity column in fraction 8 (Right), with a low molecular weight δ60ym-binding activity, most probably a smaller subunit of PYR complex that still retains DNA-binding activity, eluting in earlier fractions (peak in fraction 6). Bands with approximate molecular masses of 48, 55, and 150 kDa (p48, p55, and p150, arrows) from pooled fractions 7–9 subsequently were identified as SWI/SNF complex subunits INI1 (p48) BAF57 (p55), and BAF155 (p150) by mass spectrometric analysis.