Purification of neutravidin from E. coli lysate by batch affinity chromatography with AVD-A1(Resin)


Overview
Abstract
E.coli were lysed with BPER (Thermo), cleared by centrifugation, and diluted with PBS to 1.2 mg total protein/ml. The lysates were spiked with Avidin or Neutravidin to 0.1 mg/ml in a volume of 1.4 ml and incubated with 10 µl (packed vol) AVD-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
Both avidin and neutravidin were successfully purified to near homogeneity from spiked E.coli lysates.
Figure

Image 

Neutravidin purification with nanoCLAMP AVD-A1 affinity chromatography

Legend 

SDS-PAGE analysis of one step purification of Avidin and Neutravidin from E.coli BL21DE3 whole cell lysates using AVD-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 lysates were spiked with Avidin or Neutravidin to 0.1 mg/ml in a volume of 1.4 ml and incubated with 10 µl (packed vol) AVD-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.

Lanes M: 10 µl See Blue Plus pre-stained protein marker (Thermo).

Lane 1: Avidin-spiked lysate containing 21 ug total protein.

Lane 2: 10 µl of buffer exchanged Avidin polyol eluant from the affinity resin, from a total of 130 µl total elution. 

Lane 3: Neutravidin-spiked lysate containing 21 µg total protein.

Lane 4: 10 µl of buffer exchanged Neutravidin polyol eluant from the affinity resin, from a total of 131 µl total elution.