[Introduction]

RuBisCo is an enzyme that is used in Calvin cycle to catalyze the first major step of carbon fixation, a process by which the atoms of atmospheric carbon dioxide are made available to organisms in the form of energy rich molecules such as sucrose. It constitutes about 40% of total plant proteomics and thus interferes with proteomics studies. The RuBisCo Seppro® Tip is specifically designed to remove RuBisCo protein from plant extract. It is based on avian antibody (IgY)-antigen interactions and optimized buffers for sample loading, washing, eluting and regenerating. The targeted RuBisCo protein are simultaneously removed by the immobilized specific IgYs when crude biological samples are passed through the tip. Selective immunodepletion provides an enriched pool of low-abundant proteins for downstream proteomics analysis. Specific removal of RuBisCo protein depletes about 40% of total protein mass from green leaves. The low-abundant proteins in the flow-through fractions can be studied. Removal of RuBisCo protein enables improved resolution and dynamic range for one-dimensional gel electrophoresis (1DGE), two-dimensional gel electrophoresis (2DGE) and liquid chromatography/mass spectrometry (LC/MS). The collected flow-through fractions may need to be concentrated depending on the downstream applications.

The Seppro® Tip products are designed to use with PSS Bio Instrument’s automated Magtration® System APT12 or 12GC. Twelve tips are simultaneously operated and twelve samples can be processed at once with no hand-on manipulations in approximately 65 minutes.

[Product Description]

Seppro® Tip IgY-Rubisco and Reagent Kit

Product Name "	Quantity	Product Description
Rubisco Sepproｮ Tip (~0.5 ml packed bed)	12	Immobilized IgY antibody beads for Immunoaffinity capture of Rubisco Tip capacity: ~0.4 mg Rubisco protein
Dilution Buffer 10x	10 ml	1xTris-Buffered Saline (TBS): 10 mM Tris-HCl, pH 7.4,150 mM NaCl
Elution Buffer	10 ml	1M Glycine-HCl, pH 2.5
Pre-filled Reagent Cartridges	48	Wells 1, 2, 3, 9, 10: 0.5 ml of TBS each ; Wells 4, 5, 6, 7: elution buffer (0.5ml of 0.25 M Glycine-HCl, pH 2.5 each) Well 8: Neutralizing Buffer (0.5 ml of 0.25 M Tris-HCl, pH 8.0)
2-ml screw cap tubes	160	Plastic tubes for filling the sample (Row4 position), Wash buffer (Row 3 position) and Elution buffer (Well 11 position). Please read the specification for screw cap tubes in the instruction provided with the 12GC instrument if you would like to use your own tubes
,

Note: Additional pre-filled cartridges and bulk reagents can be ordered separately.

[Cartridge Arrangement]

Wells 1-3: Wash Buffer (TBS, 10 mM Tris-HCl, 0.15 M NaCl, pH 7.4)
Wells 4-7: Elution Buffer (0.25 M Glycine-HCl, pH 2.5)
Well 8: Neutralizing Buffer (0.25 M Tris-HCl, pH 8.0)
Wells 9-10: TBS (10 mM Tris-HCl, 0.15 M NaCl, pH 7.4)
Well 11: 2-ml tube with 2-ml of Elution buffer (0.1 M Glycine-HCl, pH 2.5) (prepared and filled by users)

[Automated Protocol]

(For the set-up of Magtration® System 12GC instrument, accessories and disposable, refer to Operation Manual)

A. Pre-treatment of Seppro® Tip---a ‘mock run’ to remove any residual non-covalently bound IgY from the beads

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. Turn on 12GC and lift the door for reagent loading.
3. Load the Reagent Cartridges into cartridge rack according to the number of the samples and tips to be used (12GC holds maximal 12 tips for 12 samples/per run).
4. Insert the cartridge rack into the rack setting position on the stage.
5. Remove the top cap of the Seppro® tip, place it at Row 2 position of the tip/tube rack together with tip sheath. (Note: Equilibriate Seppro® tip at the room temperature for 10-15 mins before performing either mock run or depletion run). No tubes are required to be setup in the tip/tube rack while performing the mock run protocol.
6. Set the tip/tube rack into the instrument.
7. Close the door and follow the instruction on the display screen of the instrument to start the protocol for the whole process. It will take about 1hour to finish the process.

B. Depletion run--- Removal of 12 abundant proteins from the human serum/plasma samples.

The setting of cartridge rack is essentially the same as for the mock run protocol described above for the mock run protocol except that in well 11 position of the cartridge rack insert a 2-ml tube containing 2-ml of elution buffer.

The setting of the Tip/tube rack is as follows:

Row 4: Unfractionated sample, 2 ml screw cap tube
Row 3: 2 ml tube with 2-ml of Wash buffer (10 mM Tris-HCl, 0.15 M NaCl, pH 7.4) (prepared and filled by users only for depletion run) Row 2 : Seppro® Tip with the Tip-holder
Row 1: Empty

Close the door and follow the instructions on the display screen to start the depletion protocol. It will take about 1 hour to finish the process. Research use only Ver.4_092507

Note: At the end of the depletion protocol fractionated serum sample, wash and elution fractions will end up in the following rows of Tip/tube rack and Wells of reagent cartridge. Collect the fractions into the tubes for further analysis.

Plasma or serum sample (250 ul or 500 ul), Row 4 (Tip/Tube rack) Wash fraction 1 (~500 ul), Well 1 (Cartridge)
Wash fraction 2 (~500 ul), Well 2 (Cartridge)
Wash fraction 3 (~500 ul), Well 3 (Cartridge)
Wash fraction 4 (~500 ul), Row 3 (Tip/Tube rack)
Elution fraction 1(~500 ul) , Well 4 (Cartridge)
Elution fraction 2(~500 ul), Well 5 (Cartridge)

C. Sample preparation and processing

1. Centrifuge human serum/plasma samples at 10,000 rpm for 1 min and use the clear supernatant for processing. Alternatively, use Spin X filters (Fisher, 0.45 um) to remove any particles from human serum/ plasma by centrifuging at 10, 000 rpm for 1 min.
2. Dilute a sample properly using TBS to get a volume of either 250 ul or 500 ul containing about ~0.4 mg Rubisco in a 2-ml screw cap tube.
3. Load the sample tube in the Row 4 of the cartridge Tip/tube rack and start the protocol for sample processing.
4. After completing the procedure, neutralize the eluted fractions by adding 200 µl of 1M Tris-HCl into each tube at positions -4 and 11. 5. Store the tip with Dilution Buffer at 2–8°C in a refrigerator. For long term storage, use Dilution Buffer with 0.02% sodium azide. DO NOT FREEZE THE TIPs.
6. It is desirable to start a ‘mock run’ before a consecutive depletion runs to give a constant capacity of the Tip.

[Seppro®Tip specifications]
Tip Size:

Tip body materials:

Column capacity:

Operating temperature:

Immobilized ligands:

Shipping solution Buffer:

Shipping temperature:

Storage temperature:

[Safety Issues]

When preparing biological samples using Seppro® Tip IgY-12, follow general guidelines for handling biological materials and wear protective eyewear and gloves.

[Recommendations]

Sample dilution
Dilute the sample properly in dilution buffer to the final volume containing about ~0.4 mg Rubisco.

Sample cleanup
To avoid column clogging it is recommended to filter the sample using a 0.45-µm spin filter.

Tip performance
Always follow the instruction.

Tip storage
Always store the Tips after equilibrating with Dilution Buffer in a refrigerator at 2–8°C when not in use to minimize loss in Tip capacity.

Caution: Do not expose Tips to organic solvents (like alcohols, acetonitrile, etc.), strong oxidizers, acids, or reducing agents, and other protein denaturing agents.

Warning: FOR RESEARCH USE ONLY - this product is NOT TO BE USED AS AN IN-VITRO DIAGNOSTIC.

Storage: Store the affinity column at 2-8°C upon receiving and when not in use. DO NOT FREEZE THE TIP! Store tips with the end-caps tightly sealed.

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

Seppro® is a registered mark owned by GenWay Biotech, Inc. 6777 Nancy Ridge Dr., San Diego, CA 92121, USA TEL: 858-458-0866, FAX: 858-458-0833 http://www.genwaybio.com ------------------------------------------------------------------------------------------- Precision System Science Co., Ltd.
88, Kamihongou, Matsudo-shi, Chiba, 271-0064, Japan
TEL: +81-47-303-4800, FAX: +81-47-303-4811
http://www.pss.co.jp

Precision System Science Europe, GmbH
Spiesheimer Weg 32
D-55286 Wörrstadt
Germany
TEL : +49 6732 9388 0, FAX :+49 6732 9388 20
http://www.pss-europe.de

Precision System Science USA, Inc.
6052 Industrial Way, Suite H, Livermore, CA 94551, USA
TEL: 925-960-9180, FAX: 925-960-9184
http://www.pssbio.com

[Technical Support]

Precision System Science USA, Inc.
6052 Industrial Way, suite H, Livermore, CA 94551, USA
Tel: 925-960-9180, Fax: 925-960-9184
E-mail: contact@pssbio.com
http://www.pssbio.com