NusA purification by affinity chromatography with nanoCLAMP nusA-A1(Resin), batch mode from E. coli lysate


Overview
Abstract
NusA purification by affinity chromatography with nanoCLAMP nusA-A1(Resin) in batch mode from E. coli lysate. E.coli were lysed with BPER (Thermo), cleared by centrifugation, and diluted with PBS to 1.2 mg total protein/ml. The lysate was spiked with recombinant antigen to 0.1 mg/ml in 1.4 ml and incubated with 10 µl (packed vol) nusA-A1(Resin). Beads were washed with PBS and eluted with polyol elution buffer at near neutral pH.
Type
  • Affinity Chromatography
Target
Scores
Outcome
Successful
Signal
Acceptable
Cross Reactivity
Acceptable
Background
Acceptable
Dynamic Range
Acceptable
Results
Summary
NusA was successfully purified from an E.coli whole cell lysate to apparent homogeneity in one chromatography step.
Figure

Image 

NusA purification by nanoCLAMP nusA-A1 affinity chromatography

Legend 

SDS-PAGE analysis of one step purification of NusA from E.coli BL21DE3 whole cell lysates using nusA-A1(Resin) affinity resin. E.coli were lysed with BPER (Thermo), cleared by centrifugation, and diluted with PBS to 1.2 mg total protein/ml. The lysate was spiked with recombinant antigen to 0.1 mg/ml in 1.4 ml and incubated with 10 µl (packed vol) nusA-A1(Resin). Beads were washed with PBS and eluted with polyol elution buffer at near neutral pH. Lysates and eluants were analyzed on 12% SDS-PAGE in reducing SDS sample buffer.

Lane 1: NusA-spiked lysate containing 10 ug total protein.

Lane 2: 6.5 µl of polyol eluant from the affinity resin, from a total of 125 µl total elution. Note: the band in lane 2 at around 40 kDa was analyzed by mass spec and found to be a fragment of NusA.