Beta-galactosidase purification by affinity chromatography with nanoCLAMP lacZ-A2(Resin), batch mode from E. coli lysate


Overview
Abstract
Beta-galactosidase purification by affinity chromatography with nanoCLAMP lacZ-A2(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) lacZ-A2(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
Beta-galactosidase was successfully purified from an E.coli whole cell lysate to apparent homogeneity in one chromatography step.
Figure

Image 

SDS PAGE of lacZ purification with nanoCLAMP lacZ-A1 affinity chromatography

Legend 

SDS-PAGE analysis of one step purification of beta-galactosidase from E.coli BL21DE3 whole cell lysates using lacZ-A2(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) lacZ-A2(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: Beta-galactosidase-spiked lysate containing 10 ug total protein.

Lane 2: 8 µl of polyol eluant from the affinity resin, from a total of 125 µl total elution.