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


Overview
Abstract
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) trxA-A1(Resin). Beads were washed with PBS and eluted with polyol elution buffer at near neutral pH.
Type
  • Affinity Chromatography
Specimen
E. coli Whole Cell Extract
Target
Thioredoxin-1 (E. coli)
Scores
Outcome
Successful
Signal
Acceptable
Cross Reactivity
Acceptable
Background
Acceptable
Dynamic Range
Acceptable
Results
Summary
Thioredoxin fusion protein was successfully purified from an E. coli whole cell lysate to apparent homogeneity in one chromatography step.
Figure

Image 

SDS PAGE thermoredoxin purification with nanoCLAMP trxA-A1 affinity chromatography

Legend 

SDS-PAGE analysis of one step purification of a Thioredoxin-fusion protein from E. coli BL21(DE3) whole cell lysates using trxA-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) trxA-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: Thioredoxin-spiked lysate containing 21 µg of total protein.

Lane 2: 16 µl of polyol eluant from the affinity resin, from 127 µl total elution.
Methods
Protocol
Affinity purification from E.coli lysates using nanoCLAMP(Resin)
Product Lot(s) Used
Lot Function Academic Price Commercial Price Order Equivalent Product
nC-trxA-A1(Resin)750ul-06 Capture $475 $975
Conjugate(s) Used
ConjugateFunction
nanoCLAMP trxA-A1(Resin)
  • Capture