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ReadyPrep

™

2-D Cleanup Kit

Instruction Manual

Catalog #163-2130

For technical service, call your local Bio-Rad office, or

in the US, call 1-800-4BIORAD (1-800-424-6723)

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Summary of content (20 pages)

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4110143A4.qxd 1/8/2003 6:08 PM Page 2 Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
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4110143A4.qxd 1/8/2003 6:08 PM Page 3 Table of Contents Section 1 Introduction……………………………………...1 Section 2 Kit Specifications…………………………......2–3 Section 3 Storage Conditions………………………..........3 Section 4 Instructions for Use………………….............4–9 Section 5 Appendix……………..……………............10–15 Section 6 Product Information………………….........
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110143A4.qxd 1/8/2003 6:08 PM Page 4 Section 1 Introduction The ReadyPrep 2-D cleanup kit facilitates the preparation of low conductivity samples suitable for isoelectric focusing (IEF) and 2-D gel electrophoresis. Additionally, the kit concentrates proteins from samples that are too dilute, allowing for higher protein loads that can improve spot detection.
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4110143A4.qxd 1/8/2003 6:08 PM Page 5 Section 2 Kit Specifications Each ReadyPrep 2-D cleanup kit provides sufficient reagent to purify 50 samples of 100 µl, each sample containing up to 500 µg of protein. The kit contains the components listed below. Certificates of analysis or MSDS forms are available upon request.
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4110143A4.qxd 1/8/2003 6:08 PM Page 6 Items Required But Not Provided: • 1.5 ml microcentrifuge tubes • Microcentrifuge capable of spinning at 12-16,000 x g at 4°C. • Rehydration/sample buffer (see Section 5 for recipes and Section 6 for premixed, ready-to-use rehydration/sample buffers) • Vortex mixer • ReadyPrep proteomic grade water or other ultrapure water • RC DC™ Protein Assay (Bio-Rad catalog #500-0121 or #500-0122) Section 3 Storage Conditions Store the kit at room temperature.
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4110143A4.qxd 1/8/2003 6:08 PM Page 7 Section 4 Instructions for Use 4.1 Protocol for Sample Volumes up to 100 µl Use 1.5 ml microcentrifuge tubes for processing the protein samples. Maintain the sample tubes in an ice bucket during each step of the procedure unless otherwise specified. Always position the microcentrifuge tubes in the centrifuge in the same orientation (for example: cap hinge facing outward) so that the protein pellet remains on the same side of the tube after each spin.
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4110143A4.qxd 1/8/2003 6:08 PM Page 8 1. Transfer 1–500 µg of protein in a final volume of 100 µl into a 1.5 ml microcentrifuge tube. Note: Sample quantities > 500 µg of protein may reduce the efficiency of the cleanup leading to poor-quality IEF. 2. Add 300 µl precipitating agent 1 to the protein sample and mix well by vortexing. Incubate on ice for 15 min. Note: When adding solution, do not touch protein sample with the pipet tip. The protein may precipitate on the tip causing sample loss. 3.
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4110143A4.qxd 1/8/2003 6:08 PM Page 9 bottom of the tube. Use a pipet to carefully remove the remaining supernatant. 7. Add 40 µl of wash reagent 1 on top of the pellet. Position the tube in the centrifuge as before and centrifuge at maximum speed (> 12,000 x g) for 5 min. Note: A precipitate may form along the tube wall. In these cases, vortex and/or pipet wash solution over the pellet several times to ensure entire pellet is thoroughly washed. 8. With a pipet, remove and discard the wash. 9.
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4110143A4.qxd 1/8/2003 6:08 PM Page 10 discard the supernatant. Centrifuge the tube briefly (15–30 sec) and remove and discard any remaining wash. The pellet will appear white at this stage. Air-dry the pellet at room temperature for no more than 5 min (the pellet will look translucent once sufficiently dry). Note: Do not over-dry pellets. Over-dried pellets will be difficult to resuspend. 13. Resuspend each pellet by adding an appropriate volume of 2-D rehydration/sample buffer to the pellet.
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4110143A4.qxd 1/8/2003 6:08 PM Page 11 At the end of the process, some pellet fragments may remain undissolved. When using a urea/thiourea-based 2-D rehydration/sample buffer, all the protein is extracted from the pellet if the above protocol is followed. 14. Centrifuge the tube at maximum speed for 2–5 min at room temperature to clarify the protein sample. The supernatant can be used directly for IEF in IPG strips or in IEF gels.
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4110143A4.qxd 1/8/2003 6:08 PM Page 12 reduce possible spilling during vortexing and the incubation at -20°C. A Nalgene Oak Ridge-type tube made of polypropylene copolymer is an example of an appropriate tube. The volume of the tube should be at least 12 times the original volume of the sample. Step 2. For each volume of sample use 3 volumes of precipitating agent 1. Step 3. For each volume of sample use 3 volumes of precipitating agent 2. Step 7.
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4110143A4.qxd 1/8/2003 6:08 PM Page 13 Section 5 Appendix 5.1 2-D Rehydration/Sample Buffer Volume In the final step for this kit, all samples are resuspended in a 2-D rehydration/sample buffer (Section 5.2). To best determine the volume of 2-D rehydration/sample buffer to use, consider the questions listed below.
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4110143A4.qxd 1/8/2003 6:08 PM Page 15 5.2 Preparation of 2-D Rehydration/Sample Buffers The 2-D rehydration/sample buffer is not a component of this kit, but is necessary for resuspending the final protein pellet. It is not provided in the kit because different protein samples can require different rehydration/sample buffers. For convenience, a selection of 2-D rehydration/sample buffer formulas are provided below.
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4110143A4.qxd 1/8/2003 6:08 PM Page 16 5.2.1. General-Purpose 2-D Rehydration/Sample Buffer for Most Samples. (8 M Urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte  3/10 ampholyte, 0.002% bromophenol blue) Component Final Concentration Amount to Make 2 ml Urea (FW 60.06) CHAPS DTT (FW 154.3) 100X Bio-Lyte 3/10 ampholyte* Bromophenol Blue 8M 2% (w/v) 50 mM 0.2% (w/v) 0.96 g 0.04 g 15.4 mg 20 µl 0.002% (w/v) 4 µl of a 1% (w/v) solution 1.
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4110143A4.qxd 1/8/2003 6:08 PM Page 17 5.2.2. Strongly Chaotropic 2-D Rehydration/Sample Buffers for Highly Hydrophobic Proteins. 5.2.2.1. Standard Strongly Chaotropic Buffer (7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 0.2% Bio-Lyte 3/10 ampholyte, 0.002% Bromophenol Blue). Component Final Concentration Amount to make 2 ml Urea (FW 60.06) Thiourea (FW 76.12) CHAPS* DTT (FW 154.3) 100X Bio-Lyte 3/10 ampholyte** Bromophenol Blue 7M 2M 4% (w/v) 50 mM 0.2% (w/v) 0.84 g 0.304 g 0.08 g 15.4 mg 20 µl 0.
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4110143A4.qxd 1/8/2003 6:08 PM Page 18 5.2.2.2. Protein Solubilization Buffer (PSB). Protein solubilization buffer (PSB) and PSB diluent are provided with ReadyPrep protein extraction kit (membrane I) and ReadyPrep protein extraction kit (cytoplasmic/nuclear) to solubilize the protein pellet after using the ReadyPrep 2-D cleanup kit. PSB is a proprietary, strongly chaotropic 2-D rehydration/sample buffer that will solubililze both hydrophilic as well as hydrophobic proteins.
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4110143A4.qxd 1/8/2003 6:08 PM Page 20 Buffer Components 161-0611 163-2101 161-0460 161-0731 161-0716 161-0302 163-2094 163-2091 Dithiothreitol (DTT), 5 g Tributylphophine (TBP), 200 mM, 0.6 ml CHAPS, 1 g Urea, 1 kg Tris, 500 g Sodium Dodecyl Sulfate (SDS), 1 kg 100X Bio-Lyte 3/10 Ampholyte, 1 ml ReadyPrep Proteomic Grade Water 2-D Rehydration/Sample Buffers 163-2106 ReadyPrep 2-D Starter Kit Rehydration/Sample Buffer, 10 ml, containing 8 M urea, 2% CHAPS, 50 mM DTT, 0.