[Introduction] Magtration® is a proprietary technology developed by PSS to perform magnetic separation in a pipette tip similar to filtration. A series of compact Magtration® robots have been developed for automated purification of nucleic acids and proteins using reagents pre-filled in sealed cartridges. Magtration-His^TM Cartridge-WS is developed for automated purification of His-tag water-soluble proteins on the Magtration® 12GC robot. Up to 12 x 10 ml samples can be processed in parallel on the robotic system in 30 min. Ni2+ or Co2+ magnetic beads used for the purification can be recharged by automation on the same cartridge containing pre-filled reagents for both protein purification and bead recharging.

[Magtration-His^TM Kit contents]
Cartridge-WS 48
Tip/Sheath sets 50
1.5-ml Micro Tubes 150
SureLyse^TM reagent 55 ml
Ni2+ charge solution 40 ml
[Storage Condition]
Do not store
the cartridges upside down.
Do not freeze them.
Store the reagents at the
room temperature.
Avoid high temperature
and humidity.

[Preparation of bacterial lysate samples]
Please refer to [Bacterial Lysate Preparation using SureLyse™ Reagent] (Supplemental sheet, SP001). This lysis method has been developed to ensure effective lysis of bacterial cells for the protein purification.

[Ni2+/Co2+ magnetic beads from different vendors]
Please refer to [Use of Ni2+ /Co2+ Magnetic beads for Magtration-His™ kits] (Supplemental sheet, SP002). Users need to purchase Ni2+ or Co2+ magnetic beads from different vendors, and select a suitable amount of the beads corresponding to the protein expression level.

Please read the Magtration® 12GC User Manual before starting operation on the robot.

[Set up for the cartridges and consumables]
1. Make sure all the buffers in the cartridge are on the bottom of each well. If not, gently tap the cartridge on a lab bench.
2. Insert the cartridge into a cartridge rack, and then,
3. Insert a 1.5-ml tube containing Ni2+ or Co2+ charge solution into Well #11 of the cartridge for bead recharging after purification.
4. Place the cartridge rack in the instrument.
5. Set up tips and tubes in a tip/tube rack:

Row 4, 1.5-ml tube with sample, Final volume: 600 µl for 1-3 ml pellet 1200 µl for 4-10 ml pellet (cf. Supplemental sheet, SP001)
Row 3, Tip in a tip sheath
Row 2, 1.5-ml tube with beads, re-suspended in ultrapure water to a final volume of 800 µl. (cf. Supplemental sheet, SP002)
Row 1, Empty 1.5-ml tube (Eluate)

6. Place the tip/tube rack into the instrument.

[Operation procedures]
1. Make sure the power is OFF for the robot, and then insert a Magtration-His^TM protocol IC card into the inlet of the robot.
2. Turn on the power,
a. Confirm all consumables, magnetic beads and samples are set correctly as shown above, and remove caps from the all tubes.
b. Close the front door of the robot, and press “START” key to follow the on-screen instructions to select the protocol and options to start an automated purification run.

[Selection of protocols and options for robotic runs]

1. Select protein “Purification” protocol from menu screen, and follow screens with the options:
(i) Sample size: “1-3 ml pellet” or “4-10 ml pellet”,
(ii) Imidazole concentration: 0, 5, 10 or 20 mM,
(iii) Elution volume: 150 µl or 250 µl (only for 4-10 ml protocol),
(iv) Start a purification run (approx. 30 min).
(v) When the run is completed, a beep sounds, open the door and collect the elution tubes containing proteins.

3. Select “Bead regeneration after purification” protocol from menu screen, and users need to check and insert a tube containing Ni2+ or Co2+ charge solution into Well #11, and press any key to start the bead recharging run (approx. 40 min).
Notes: The beads are suspended in Well #10 of the cartridge after the purification, and replaced back into the bead tube only after the recharging, and they are ready to be reused for a next purification run with the same protein. The bead regeneration protocol is used only after the purification with the pierced cartridges.

Instructions to select different protocol and options from 12GC operation panel are shown on the back side of this sheet

**[Troubleshooting]**
Symptoms	Possible solutions
High background (lower purity)	a.Reduce amount of magnetic beads. b.Select 10 or 20 mM imidazole conc.in the wash buffer. c.Try magnetic beads with a different divalent cation. d.If the His-tag protein is expressed at a very low level, optimize expression.
Purified protein is degraded	a.Include protease inhibitors in the lysis buffer when lysozyme treatment is used. b.If prepared by sonication, lyse the cells on ice in presence of protease inhibitors.
Low purification yield for His-tagged proteins	a.Optimize the induction to increase the protein expression. b.Verify that the lysis method is sufficient. For lysozyme treatment it is recommended to use freshly prepared lysozyme. c.Reduce the amount of imidazole used. Select ・・or ・ mM・imidazole concen-tration from the menu screen to run the purification protocol. d.Increase the amount of beads if the flow-through fraction is shown to contain a significant amount of the protein of interest. e.The polyhistidine tag may not be accessible on the surface of the protein. Change the location of the polyhistidine-tag in the DNA sequence. f. Polyhistidine tag may be missing. Verify by DNA sequencing.

(1) When TOP MENU appears, press START key from the operation panel and this screen briefly flashes...

(2) Press “1” to select an automated protocol for His-tag protein purification.

(3) Press “1” or “2” to select sample size of 1-3 ml pellet or 4-10 ml pellet, respectively. A corresponding screen panel will appear for confirmation, press START to continue…
(4) Press “1” to select 0 mM imidazole to the sample and wash buffer. Press “2, 3 or 4” to select a different imidazole concentration in the wash buffer, and the concentration is adjusted to be always 10 mM in sample tube.
(5) For a sample size of 4-10 ml pellet, there is an option to select a different elution volume. For the sample size of 1-3 ml pellet, the elutiovolume is set at 150 µl. Press "ESC" key to
go back to the previous
screen or any other
key to start...

At the end of the purifiation,
TOP MENU appears again,
then Press START

(1) Press "2" to select
regeneration of the
magnetic
beads, which are already
dispensed in a well of the
cartridge.

(2)This screen appears to
remind users to check for
Well #11
if they have placed a tube
containing Ni²⁺ or Co²⁺
change solution.

Press "ESC" key to go
back to
the previous screen or any
other key to start...

Magtration® is a registered mark owned by Precision System Science Co., Ltd.

[Manufactured and sold by]
Precision System Science Co., Ltd. 88, Kamihongou, Matsudo-shi, Chiba, 271-0064, Japan TEL: +81-47-303-4800, FAX: +81-47-303-4811

PSS USA, Inc. 6052 Industrial Way, Suite H, Livermore, CA 94551, USA TEL: 925-960-9180, FAX: 925-960-9184

[Technical Support]
PSS USA, Inc. 6052 Industrial Way, suite H, Livermore, CA 94551 Tel: 925-960-9180 E-mail: contact@pssbio.com http://www.pssbio.com