Editing Protein Purification using Magnetic Beads (section)

= His-tag protein purification using Ni-NTA magnetic beads<br><br> =

== Buffers ==

'''Extraction buffer:'''

20 mM Tris-HCl, pH 8.0<br> 1 mM PMSF<br> 1 mg/ml lysozyme<br> 0.05% Triton X-100

'''Magnetic PBS:'''

10 mM potassium phosphate, pH 7.2<br> 250 mM NaCl<br> 0.05% Triton X-100

'''Washing buffer:'''

10 mM KP, pH 7.8<br> 300 mM NaCl<br> 20 mM imidazole<br> 5% glycerol<br> 0.2% Triton X-100

== Materials: ==

Ni-NTA magnetic beads (Qiagen)<br> 1.5-ml microfuge tubes<br> sample mixer equiped with tube-holding wheel (Dynal)<br> magnetic rack (Dynal MPC-S)<br> Ultrasonic water bath (Branson 200)

== Preparation of the magnetic beads: ==

Pipette 30 µl of the magnetic suspension from the bottle and apply it to a 1.5-ml microfuge tube.<br> Place the tube in the magnetic rack, apply the magnet and remove the supernatant.<br> Wash the beads with 300 µl magnetic PBS.<br> Resuspend the beads in 50 µl extraction buffer.

== Procedure: ==

Apply 1.3 ml of bacterial culture to a microfuge tube and spin for 5 min at 13,000 rpm. Store the pellet at -20°C<br> Add 300 µl of extraction buffer to the pellet, resuspend the cells and incubate the suspension for 30 min at room temperature.<br> Homogenize by sonicating the cell suspension for 3 min in the ultrasonic water bath.<br> Take 10 ml of the homogenate and store for SDS-PAGE analysis. Centrifuge the rest for 10 min at 13,000 rpm.<br> Remove the supernatant and discard the pellet. Take a 10-µl sample of the supernatant for SDS-PAGE analysis.<br> Pipette the rest of the supernatant to the prepared magnetic beads (in a 1.5-ml microfuge tube) and incubate the tube in the rocking device for 30 min at room temperature.<br> Apply the magnet and remove the supernatant.<br> Wash the beads with 300 µl washing buffer for 30 min at room temperature using the rocking device.<br> Then, wash the beads with 300 µl of PBS for 10 min.<br> Pipette 12 µl SDS sample buffer to the beads and boil for 3 min.<br> Mix the homogenate and supernatant samples (10 µl) with 10 µl SDS sample buffer and boil for 3 min.<br> Analyze the samples [homogenate (total protein), supernatant (soluble protein) and purified protein] by SDS-PAGE.

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== High-throughput Protocol<br> ==

=== 1. Grow Bacteria Overnight in 2 ml of Autoinduction Medium Using 24-well Plates ===

Cells: lac-inducible BL21 (DE3) Rosetta 2 cells<br>

Sterilise the 24-well plates by microwaving.<br>

<br> Inoculate 1.5 ml of autoinduction medium with freshly grown bacterial colonies or frozen glycerol stocks. <br>

Usually inoculate 2 wells per clone with 2 individual cloned colonies.<br>

Reserve 4 wells for positive (His-MBP) and 1 negative control (transformed with non-expressing plasmid)<br>

Check for sufficient sterilization of the plate by leaving one well blank for the medium-only control.<br>

Grow the cells for 18 hr at 37 °C and shaking at 250 rpm. <br>

Autoinduction medium contains a mixture of glucose and lactose. The bacteria initially feed on glucose and then begin to use lactose, which also induces the expression of the recombinant proteins.<br>

=== Buffers ===

Make up the Buffers<br>

'''Wash buffer '''<br>

20 mM imidazole, 0.1% Triton X-100, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9,&nbsp; 10% glycerol<br>

'''Elution buffer'''<br>

0.3 M imidazole, 0.1 % Triton X-100, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9,&nbsp; 10% glycerol. <br>

These buffers will be used to wash the beads after the tagged proteins have been bound to them, or to elute the proteins from the beads, respectively.<br>

<br> Make up the lysis solution consisting of 10x FastBreak reagent, 2 μl lysonase per sample and 15 mM MgOAc2. FastBreak contains a mix of detergents and salts that break the bacterial cell walls and facilitate the release of intracellular proteins. Lysonase is a proprietary mix of lysozyme and a nuclease. Lysozyme assists in disrupting the cell wall and the nuclease digests the released bacterial nucleic acids.<br>

<br> Optional: Make up a 6 M guanidine hydrochloride solution. Some recombinant proteins tend to stick to the Teflon-coated pipetting needles. The guanidine hydrochloride solution denatures proteins and washes them off more effectively than water that is routinely used to rinse the washable robotic pipetting tips after each step.<br> <br> '''Lysis, Binding, Washing and Elute'''<br>

The Cells are Broken up to Release the Proteins and to Allow Bead-binding

MagneHis Ni-Particles:&nbsp; Promega V8565<br>

100 μl of magnetic Ni-bead suspension is distributed into each well of a second 24-well plate.<br> 900 μl of each small-scale bacterial culture is transferred into the 24-well plate containing the beads. Add 100 μl of the 10x FastBreak/lysonase mix.<br> The 24-well plate is transferred to the shaking platform for rapid shaking (800 rpm) for 30 min at room temperature. Bacterial cell walls are disrupted by a combination of mechanical forces and chemical action and the recombinantly produced proteins are released into solution. With their affinity tags they immediately bind the paramagnetic chelating nickel beads.

The cell-lysates containing the resuspended magnetic beads are transferred to a 96-deepwell plate which is positioned on a stand with magnetic rods that slide between the wells. The 96-well plates have got a square cone-shaped bottom which allows easier removal of the supernatant The wells can hold volumes of up to 2.1 ml but are filled with much smaller volumes. This enables us to perform vigorous shaking steps without sample cross-contamination by splashing.<br>

<br> The pipette tips are washed between individual pipetting steps with 6 M guanidine-HCl. This step is crucial for the purification of&nbsp; some proteins to avoid cross-contamination. <br>

<br> The magnetic rods of the magnetic stand attract the paramagnetic beads, pulling them away from the centres of the wells and allow the pipetting needles free access to the supernatant. The supernatant is discarded.<br>

<br> 500 μl of wash buffer is first added to the 24-well plate and subsequently transferred to the 96-well plate to ensure the complete transfer of the beads. The 24-well plate is removed from the shaker.<br>

<br> Another 500 μl of wash buffer is added directly to the 96-well plate, the plate is transferred to the shaker and vigorously shaken for 1 min. The plate is moved back to the magnetic stand and the wash buffer discarded.<br>

<br> This step is repeated twice and the wash procedure is finished by removing any buffer remnants from the plate.



'''Elute'''<br>

100 μl of elution buffer is added to the beads, the plate is moved to the shaker and vigorously shaken for 30 min at room temperature.<br> The plate is moved back to the shaker and the eluate, containing the purified recombinant protein, is transferred to a new plate.

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Measure the Concentrations of the Purified Proteins<br><br>

FastBreak Promega Ltd. V8573 <br>Lysonase Bioprocessing Reagent/ 1 ml Merck Chemicals Ltd 71230 <br>Antifoam 204 Sigma-Aldrich Company Ltd A6426<br>

Deep Well Plate 2.2 ml Square Wells PP pk10 Anachem Ltd 1810-00 <br>Microplate MicroWell 96 well flat bottom polystyrene not treated 12 sleeves of 5 plates clear 0.4 ml well volume 128 mm x 86 mm Thermo Scientific Nunc Fisher Scientific Ltd. DIS-984-090M <br>Microplate Blue VWR NUNC367001 <br>24-Well Blocks RB (24) Qiagen 19583<br>

Microplate Shaker Variomag Teleshaker Inheco 3800047 <br>96-well Magnet Type A Qiagen 36915<br>

== Ref<br> ==

[http://www.jove.com/video/4110/high-throughput-purification-of-affinity-tagged-recombinant-proteins http://www.jove.com/video/4110/high-throughput-purification-of-affinity-tagged-recombinant-proteins]<br>

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[[Category:Protocols]]

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