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


Overview
Abstract
GFP purification by affinity chromatography with nanoCLAMP GFP-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) GFP-A1(Resin). Beads were washed with PBS and eluted with polyol elution buffer at near neutral pH.
Type
  • Affinity Chromatography
Specimen
Target
Scores
Outcome
Successful
Signal
Acceptable
Cross Reactivity
Acceptable
Background
Acceptable
Dynamic Range
Acceptable
Results
Summary
GFP was successfully purified from an E.coli whole cell lysate to apparent homogeneity in one chromatography step.
Figure

Image 

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

Legend 

SDS-PAGE analysis of one step purification of GFP from E.coli BL21DE3 whole cell lysates using GFP-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) GFP-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 M: 10 µl See Blue Plus pre-stained protein marker (Thermo).

Lane 1: GFP-spiked lysate containing 21 µg of total protein.

Lane 2: 16 µl of polyol eluant from the affinity resin, from 108 µl total elution.