SUMOstar affinity purification with nanoCLAMP SMT3-A1 by batch mode from over-expressing E. coli lysate.
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SDS-PAGE analysis of one step purification of SUMOstar from E. coli BL21DE3 whole cell lysate using SMT3-A1(Resin) affinity resin. E. coli overexpressing SUMOstar were lysed by sonication, cleared by centrifugation, and incubated with 1.8 ml (packed vol) SMT3-A1(Resin) for 1 h at 4°C. Beads were washed with PBS and eluted with polyol elution buffer. The eluate was buffer exchanged into Mops buffered saline, pH 7.4. Lysates and eluates were analyzed on 12% SDS-PAGE in reducing SDS sample buffer.
Lane 1: Lysate (16 ul) containing overexpressed SUMOstar.
Lane 2: Flow-through (16 ul) post-incubation on 10 ul SMT3-A1(Resin)
Lane 3: 10 ug of buffer-exchanged eluate from SMT3-A1(Resin), from a total of 6.76 mg total elution.
This protocol was scaled up to accommodate lysate from 100 ml E. coli expression culture.