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Protein purification

Dynabeads His-tag isolation and purification

Overview

Source - 40 mg/mL beads in 20% ethanol - Yield: 40 ug of 28 kDa proteins per 25 uL of beads - Sample requirements: - Free of: - EDTA (and other chelators) - Ionic detergents - DTT or DTE - pH 7-8

Buffer preparation

  • 2X Binding/Wash Buffer:
    • 100 mM Na3PO4 (pH 8)
    • 600 mM NaCl
    • .02% Tween-20
  • His Elution Buffer:
    • 300 mM Imidazole
    • 50 mM Na3PO4 (pH 8)
    • 300 mM NaCl
    • 0.01% Tween™-20

1M sodium phosphate, pH: 8.0 - Add 1.41 g of sodium phosphate dissolve in 5 ml of MiliQ water. Fill the volume up to 10 ml
- Set the pH to 8 2x Binding/Wash Buffer - 350.64 mg of NaCl - 1 ml of 1M Sodium phosphate
- 2 ul of Tween-20
- Make up volume to 10 ml with MiliQ His Elution Buffer - 175.32 mg of NaCl - 500 ul of 1M Sodium phosphate - 204.24 mg of Imidazole - 1 ul of Tween-20 - Make up volume to 10 ml with MiliQ 2x Pull-down buffer - 81.81 mg of NaCl - 65 ml of 1M Sodium phosphate - 2 ul of Tween-20 - Make up volume to 10 ml with MiliQ

Procedure

  1. Add 1X Binding/Wash Buffer to your sample.
    • The sample should be at least 100 nM or so.
    • The manual recommends 700 uL, but you can easily scale it down to 100 uL.
  2. Vortex Dynabeads for 30 s to resuspend.
  3. Transfer 50 μL (2 mg) Dynabeads to a microcentrifuge tube and place the tube on a magnet for 2 min. Discard the supernatant.
    • For small concentrations of the sample (e.g., 100 nM) can go down to 10 uL.
  4. Add your sample (in the buffer) to beads and pipette up and down.
  5. Incubate on a HulaMixer for 5 min at room temperature.
    • Might need 30 min for the incubation to be complete.
    • Can incubate the the cold room if required.
  6. Place the tube on the magnet for 2 min, then discard the supernatant.
  7. Wash the beads 4 times with 300 μL 1X Binding/Wash Buffer by placing the tube on a magnet for 2 min and discarding the supernatant.
    • Resuspend the beads thoroughly between each washing step.
    • Can use less of 1X Binding/Wash Buffer
  8. Add 100 μL His-Elution Buffer. Incubate the suspension on a HulaMixer for 5 min at room temperature.
    • Might need 30 min for the incubation to be complete.
    • Can incubate the the cold room if required.
  9. Apply on the magnet for 2 min and transfer the supernatant containing the eluted histidine-tagged protein to a clean tube.

MagStrep® Strep-Tactin®XT beads

Protocol: Protocol_MagStrep_Strep-TactinXT_Purification.pdf Tips: Protein purification with magnetic beads

Description

  • Binding capacity:
    • 42.5 ug/uL = .85 nmol/uL (assuming a 50 kDa protein)
    • For optimal yield, the total bead binding capacity should be around 5 times the total amount of protein. For example, if the total amount of a 50 kDa protein is 85 μg (= 1.7 nmol), which theoretically could be bound by 2 μl of beads (= 40 uL of 5% suspension), add 5x 2 μl = 10 μl beads = 200 uL of the 5% suspension.
    • For large proteins >90 kDa, the binding capacity may decline. To improve the yield of large proteins, increase the bead volume. Empirically, 140 kDa proteins were recovered best by 4-6X increase in the beads volume.
  • Beads volume:
    • 1 uL of the beads (=20 uL of the 5% suspension) should be mixed with a most 2.5 mL of a high-concentration sample (>1 uM).
    • Decrease the amount of beads up to 3-fold for low protein concentrations (<1 uM)

Buffers

10x Buffer W (pH 8)

  • 1 M Tris-HCl
  • 1.5 M NaCl
  • 10 mM EDTA (for antimicrobial effect; can be safely left out)

Elution Buffer BTX

  • 10X Buffer W
  • 500 mM biotin

1X Buffer P

Component Input vol. Stock conc. (M) Final conc. (mM)
Milli-Q 8.84 mL
Tris-HCl, pH 7.5-8.0 (M) 500 uL 1 50
NaCl (mM) 500 uL 3 150
MgCl2 (mM) 50 uL 1 5
Tween-20 (% v/v) 100 uL .1
DTT (mM) 10 uL 1 1
Total 10 mL
- Filter sterilize.
- (Optional): On the day of use, add RNAse inhibitor, murine (40 U/uL), ratio 1:39.
- For elution, combine 950 uL Buffer P with 50 uL 1M biotin solution.

Procedure

All steps performed at room temperature

  1. Resuspend the magnetic beads by pipetting up and down.
  2. For every 2.5 μl sample, pipette 1 μl of the 5% beads suspension into a reaction tube, place it on the magnetic separator to separate the beads and remove the supernatant.
  3. Repeat 3 times: Resuspend with 500 μl 1x Buffer W. Separate the beads in the magnetic separator and remove the supernatant.
  4. Add 250 μl sample containing the target protein and incubate for 10 minutes.
    • Keep the beads in suspension by inverting the tube occasionally, or by placing the tube on a roller.
    • Increase incubation time up to 30 min for low bead volumes.
  5. Place the reaction tube in the magnetic separator and carefully remove the supernatant.
  6. Remove tube from the magnet.
  7. Repeat 3 times: Resuspend carefully with 500 μl 1x Buffer W, place the reaction tube in the magnetic separator to collect the beads, and remove the supernatant.
  8. Elution can occur under native or denaturing conditions. Elution under native conditions offers specific elution conditions and thereby higher protein purity.
    1. Native elution: Remove the reaction tube from the magnetic separator, resuspend with 125 μl 1x Buffer BXT and incubate for 10 minutes. Place the reaction tube in the magnetic separator and transfer the supernatant containing the target protein into a new reaction tube. Repeat this step once for higher recovery. The first elution step will yield the target protein at the highest concentration.
    2. Denaturing elution: Apply conventional SDS-PAGE sample buffer and heat the sample to 95 °C for 2 minutes. Immobilized Strep-Tactin®XT will denature under these conditions and detach from the magnetic beads, leading to an additional band at 13.5 kDa during SDS-PAGE analysis. Strep-Tactin®XT magnetic beads exhibit very low non-specific protein binding activity so that no substantial amounts of other contaminating proteins should be detectable.
  9. Beads regeneration (optional):
    1. Freshly prepare the regeneration buffer (0.1 M NaOH) before use.
    2. Add 100 μl of regeneration buffer per 1 μl of magnetic beads (or 5 uL per 1 uL of 5% beads suspension).
    3. Incubate for 2 min at room temperature.
    4. Place the beads in a magnetic separator to separate them and remove the supernatant.
    5. Repeat 3 times: Equilibrate beads in 100 μl 1x Buffer W per 1 μl beads (or 5 uL per 1 uL of 5% beads suspension), separate them in the magnetic separator, and remove the supernatant.
    6. Store the beads in 1x Buffer W at 2-8 °C.