Page 42 - Macromolecular Crystallography

HIGH-THROUGHPUT CLONING, EXPRESSION, AND PURIFICATION 31

Protocol 2.3 Ni-NTA Mag bead purification in 96-well microplates

The starting material for this protocol is a deep-well 96 6. Place on the 96-well magnet for 1 min, and remove
well plate/block containing ∼1ml of E. coli expression buffer.
culture/well. The cells are first harvested by centrifugation 7. Repeat Steps 5 and 6.
at 5000 g for 15 min at 4 C, the media removed from the 8. Add 50 µl of Elution Buffer to each well, mix on the MTP
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pellets, and the resulting cell pellet frozen to −80 C for a shaker (or vortex) for 1 min, place on the 96-well magnet
minimum of 30 min (this freezing step promotes cell lysis) for 1 min, and transfer the supernatant (eluate) to a fresh
prior to use in this protocol. The protocol is adapted from MTP for analysis on SDS-PAGE. SDS-PAGE sample buffer
that used on the QIAGEN BioRobot 8000 with QIAGEN may be used instead of elution buffer if you require a more
Magnetic Ni-NTA beads but can be easily performed with concentrated sample for analysis.
the aid of a multichannel pipettor (MCP) and either a
vigorous orbital microplate shaker (minimum ‘throw’ 2 mm) Notes
or vortex mixer with MTP attachment. Bead volumes may The addition of Tween 20 to the buffers is necessary to
require adjustment if those from another supplier enable optimal collection of the magnetic beads on the
are used. sides of the MTP wells and also to facilitate efficient cell
lysis in Step 1. Bead volumes etc. are based on the
1. Resuspend the cells completely in 230 µl of Lysis Buffer QIAgen protocol and may require adjustment if reagents
supplemented with 1 mg/ml Lysozyme and either 3 units/ml from another supplier are used.
of Benzonase* (Merck, Germany; purity grade I, ≥25 U/µl, *Benzonase is recommended as it is more stable than
Cat. No. 1.01694.0001) or 400 units/ml of DNAse Type I. DNase I. When using Benzonase/DNaseI the crude
This can be done by either repeated aspirate/dispense with lysate may be used directly in the binding step but the
a suitable multichannel pipette or on an orbital microtitre results will be less informative with regards to the
plate shaker (∼1000 rpm for 30 min). Clear the lysate solubility/cellular partitioning of the proteins.
by centrifuging the deep-well block at 5000 g for 30 min
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at 4 C. Buffers
2. Approximately 5 min before the end of the Buffer NPI-10-Tween (Lysis Buffer):
centrifugation run dispense 20 µl of the Ni-NTA magnetic 50 mM NaH 2 PO 4, 300 mM NaCl, 10 mM imidazole, 1%
bead suspension (ensure full resuspension before you v/v Tween 20, adjust pH to 8.0 using NaOH and filter
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commence pipetting!) into each well of a flat-bottomed before use. Store at 4 C. 1% Tween is not required when
microtitre plate (MTP). Not all microtitre plates are magnet using Lysozyme-based lysis – 0.05% is sufficient.
compatible – check before you commence the assay. Buffer NPI-20-Tween (Wash Buffer):
3. Transfer the supernatant from Step 1 without disturbing 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole,
the ‘insoluble’ pellet to each well of the MTP containing 0.05% v/v Tween 20, adjust pH to 8.0 using NaOH and
the Ni-NTA magnetic beads. Mix for 30 min at room filter before use. Store at 4 C.
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temperature using either a MTP shaker or, alternatively, Buffer NPI-250-Tween, 50 ml: (Elution Buffer):
vortex at 600 rpm using an adapter for MTPs. The pellets 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM Imidazole,
may be resuspended in 8 M urea buffered with 100 mM 0.05% Tween 20, adjust pH to 8.0 using NaOH and
NaH 2 PO 4 and 10 mM Tris to pH 8.0 for analysis of the filter before use. Store at 4 C.
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‘insoluble’ fraction on SDS-PAGE. DNAse I Stock Solutions (40,000 units/ml):
4. Place the 96-well microplate on the 96-well magnet (we To a 200,000 unit bottle of DNAse I (Sigma D-4527) add
find the QIAgen magnets Type A or B work well but there 5 ml of UHQ sterile water. Aliquot into 25 µl aliquots
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are many other suitable magnets on the market) for 1 min store at −20 C. Make up to 25 ml with Lysis Buffer
and remove the supernatant carefully from the beads with for use.
a MCP. Lysozyme:
5. Add 200 µl of Wash Buffer to each well, remove from Weigh out 25 mg of Lysozyme (Sigma L-6876, stored at
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the magnet, and mix on the microplate shaker (or vortex) −20 C), add to the 25 ml of Benzonase/DNAse I working
for 5 min. solution described above. Use in assay immediately.