Haynes Lab:Notebook/Engineering PC-TFs/2014/09/25

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Summary

  • Purified PcTF protein.

Zymo-Spin P1 Column His-tag Purification Kit
Protein kept on ice during procedure; centrifuge in cold room used.

  • Vortexed His-Affinity Gel briefly.
  • Transferred 250μL of His-Affinity Gel to the Zymo-Spin P1 column.
  • Placed column in 2mL collection tube.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded flow-through.
  • Added 300μL PcTF in protein extraction buffer to column.
  • End-over-end mixing for two minutes to resuspend gel and allow protein to bind to resin.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded flow-through.
  • Added 250μL His-Wash Buffer and mixed to resuspend gel.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded flow-through.
  • Repeated wash step once.
  • Transferred column into a labeled 1.7mL microcentrifuge tube.
  • Added 150μL His-Elution BUffer and resuspended gel via end-over-end mixing for one minute.
  • Centrifuged for 20 seconds at max rpm.
  • Discarded column and stored purified protein at -20°C.



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