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Avidin purification by affinity chromatography with nanoCLAMP AVD-A1(Resin), batch mode with E. coli lysate
| Abstract | Avidin purification by affinity chromatography with nanoCLAMP AVD-A1(Resin) in batch mode with 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 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. |
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| Type |
|
| Specimen | |
| Target |
| Outcome | Successful |
|---|---|
| Signal | Acceptable |
| Cross Reactivity | Acceptable |
| Background | Acceptable |
| Dynamic Range | Acceptable |
| Summary | Both avidin and neutravidin were successfully purified to near homogeneity from spiked E.coli lysates. |
|---|---|
| Figure | Image LegendSDS-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 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 131 µl total elution. |