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Products >  Protein_Expression_and_Purification >  Protein_Learning_Center >  Learning_Resources >  Capturem_His-Tagged_Purification_Maxiprep_Kit_Protocol

Visual Protocol: His-Tagged Purification Miniprep Kit

VISUAL PROTOCOL

Capturem His-Tagged Purification Maxiprep Kit

This protocol provides simple, rapid purification of his-tagged proteins in up to 25 ml of clarified lysate from mammalian or bacterial cell samples using the Capturem His-Tagged Purification Maxiprep Kit (Cat. # 635713). The columns are suitable for use under native or denaturing conditions, in the presence of additives such as DTT (up to 10 mM), βME (up to 30 mM), TCEP (up to 5 mM), EDTA (up to 10 mM), or glycerol.

Pre-Purification Filtration

up to 25 ml of cleared lysate

  • Load 2–25 ml cleared lysate onto the Capturem Maxiprep Filter (purple insert)
  • Spin for 3 min
  • Discard filter
  • Save filtered lysate for Step 2

Step 1: Equilibrate

6 ml

  • Add 6 ml xTractor Buffer to the Capturem Maxiprep Nickel Column (clear insert)
  • Spin for 3 min
  • Discard flowthrough

Step 2: Bind

up to 25 ml of filtered, cleared lysate

  • Load 2–25 ml filtered lysate from the Pre-Purification Filtration step onto the Capturem Maxiprep Nickel Column
  • Spin for 3 min
  • Transfer column to a new collection tube and save flowthrough

Step 3: Wash

6 ml

  • Add 6 ml Wash Buffer* to the Capturem Maxiprep Nickel Column
  • Spin for 3 min
  • Transfer column to a new collection tube and save flowthrough

*Optimize washing conditions by adding 200–800 µl Elution Buffer to change the imidazole concentration.

Step 4: Elute

1 ml–1.5 ml

  • Add 1 ml Elution Buffer to the Capturem Maxiprep Nickel Column
  • Spin for 3 min
  • Collection tube contains your eluted tagged protein
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