Magtration® GC Automation series
Seppro® Tip IgY-R7 and Reagent Kit
Research use only
Ver. 4_092507
| [Introduction] The Seppro® Tip IgY-R7 is based on avian antibody (IgY)-antigen interactions and optimized buffers for sample loading, washing, eluting and regenerating. It is specifically designed to remove 7 high-abundant proteins from human biological fluids such as serum, plasma, and cerebral spinal fluid (CSF). This technology enables removal of Albumin, IgG, α1-antitrypsin, IgM, Transferrin, Haptoglobin and Fibrinogen in a single step. The targeted high-abundant proteins 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 fourteen high-abundant proteins depletes approximately 90% of total protein mass from human serum or plasma. The low-abundant proteins in the flow-through fractions can be studied. Removal of high-abundant proteins 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 dependent upon 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-R7 and Reagent Kit
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[Cartridge Arrangement] Wells 1-3: Wash Buffer (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 7 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 15 µl clear human serum/plasma samples using TBS to final volume of either 250 ul or 500µl 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 Well 4, 5 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: 0.5 mL bed volume Tip body materials Polycarbonate Tip, Polyethylene frits Column capacity 12-15 µL human serum/plasma Operating temperature 18–25°C Immobilized ligands Affinity-purified IgY polyclonal antibodies to human Albumin, IgG, α1-antitrypsin, IgA, IgM, Transferrin and Haptoglobin, α1-acid glycoprotein (Orosomucoid), α2 Macroglobulin, Apolipoprotein AI & AII, Fibrinogen, complement C3 and LDL Shipping solution Buffer Dilution Buffer with 0.02% sodium azide , Elution buffer Shipping temperature 18–25°C overnight or 2–8°C Storage temperature 2–8°C |
[Safety Issues] When preparing biological samples using Seppro® Tip IgY-R7, follow general guidelines for handling biological materials and wear protective eyewear and gloves. [Recommendations] Sample dilution It is not recommended to use crude serum directly. Follow instructions for plasma or serum dilution. Addition of protease inhibitors in Dilution Buffer for sample dilution helps prevent protein degradation. Sample cleanup Human serum may contain particulate materials that can be removed by a quick spin 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 |