NanoBio: Protein Purification

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Contents

His-tagged Protein Purification

Materials

  • B-PER Bacterial Protein Extraction Reagent (Fisher catalog # PI78248, Pierce catalog # 78248)
  • HisTrap FF 1 mL (Fisher catalog # 17525501)

Bacterial Lysis

  1. Follow the instructions using the B-PER reagent.
  2. Add 2M imidazole to a final concentration of 20 mM.
  3. Filter the extracted protein solution through a 0.22 um syringe filter.

FPLC affinity purification

Buffer Preparation

  • All buffers should be passed through a 0.22 um filter and allowed to equilibrate at 4C overnight before use. These steps remove particles and air bubbles which can damage the column.

Preparation of the Lines

  1. Attach the appropriate (typically 2 mL) sample loop.
  2. Rinse the sample loop with water.
    1. Select Manual: Flowpath: Injection Valve: Load.
    2. Inject 2.5 mL Millipore water into the sample injection port. Make sure that you see drops entering the waste container.
    3. Repeat 2.5 mL Millipore water injection.
  3. Replace existing buffers in lines A2 and B with Millipore water.
    1. Place lines A2 and B in ~500 mL container of Millipore water.
    2. Select Manual:
      • Pump: Flow: 5mL/min
      • Pump: Gradient: 50% B, length of gradient 0
      • Flowpath: Injection Valve: Inject
      • Flowpath: Column Position: Bypass Column Position 1
      • Flowpath: Buffer Valve: A2
      • Other: Pause TimeOut: 50 mL accumulated volume
  4. Replace the water in lines A2 and B with low imidazole and high imidazole buffer, respectively.
    1. Place lines A2 and B in low imidazole and high imidazole buffer, respectively.
    2. Select Manual:
      • Pump: Flow: 5mL/min
      • Pump: Gradient: 0% B, length of gradient 0
      • Flowpath: Injection Valve: Inject
      • Flowpath: Column Position: Bypass Column Position 1
      • Flowpath: Buffer Valve: A2
      • Other: Pause TimeOut: 35 mL accumulated volume
    3. Select Manual:
      • Pump: Flow: 5mL/min
      • Pump: Gradient: 100% B, length of gradient 0
      • Flowpath: Injection Valve: Inject
      • Flowpath: Column Position: Bypass Column Position 1
      • Flowpath: Buffer Valve: A2
      • Other: Pause TimeOut: 35 mL accumulated volume

Column Washing & Equilibration

  • Note: the maximum backpressure that a HisTrap FF column can tolerate is 0.3 MPa
  1. Equilibrate the sample loop with the appropriate buffer.
    1. Load 2.5 mL of B-PER reagent (or buffer that your protein lysate is in) in a syringe. Inject into the sample port.
  2. Wash residue off a previously used column. Select Manual:
      • Pump: Flow: 1mL/min
      • Pump: Gradient: 100% B, length of gradient 0
      • Flowpath: Injection Valve: Inject
      • Flowpath: Column Position: Column 4 (or position of HisTrap column)
      • Flowpath: Buffer Valve: A2
      • Other: Pause TimeOut: 15 mL accumulated volume (for a 5 mL column; should be ~3 column volumes)
    1. Equilibrate the column. Select Manual:
      • Pump: Flow: 1mL/min
      • Pump: Gradient: 0% B, length of gradient 0
      • Flowpath: Injection Valve: Inject
      • Flowpath: Column Position: Column 4 (or position of HisTrap column)
      • Flowpath: Buffer Valve: A2
      • Other: Pause TimeOut: 50 mL accumulated volume (for a 5 mL column; should be ~10 column volumes)

Run Programmed Separation Program

  1. Check that the fraction collector is filled with tubes and correctly aligned.
  2. Load your sample into the sample loop.
    • Select Manual: Flowpath: Injection Valve: Load.
    • Inject your sample into the sample injection port.
  3. Run CAjoHisMethod for 5 mL columns and run CAjoHisMethod1mL for 1 mL columns.
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