Manual

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ReadyPrep

™

Protein Extraction Kit

(Signal)

Instruction Manual

Catalog #163-2087

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 (18 pages)

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4110149A5.qxd 1/15/2003 2:49 PM Page 2 Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
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4110149A5.qxd 1/15/2003 2:49 PM Page 3 Table of Contents Section 1 Introduction…………………………………...1–2 Section 2 Kit Specifications…………………………......3–4 Section 3 Storage Conditions………………………..........4 Section 4 Instructions for Use………………….............5–7 Section 5 Appendix……………..……………..............8–12 Section 6 References……………..……………...............13 Section 7 Product Information………………….........
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110149A5.qxd 1/15/2003 2:49 PM Page 4 Section 1 Introduction The ReadyPrep protein extraction kit (signal) is designed as a simple, rapid and reproducible method to prepare a cellular protein fraction that is enriched in membraneassociated signaling proteins.
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4110149A5.qxd 1/15/2003 2:49 PM Page 5 Parton and Simons 1995, Anderson et al. 1992). Due to the lipid composition of these membrane subdomains, these proteins become resistant to solubilization at 4°C with many nonionic detergents such as Triton X-100 and can be selectively recovered from the detergent-insoluble membrane fraction.
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4110149A5.qxd 1/15/2003 2:49 PM Page 6 Section 2 Kit Specifications Each ReadyPrep protein extraction kit (signal) provides sufficient reagents to perform 50 extractions of 25–50 mg of cells or tissue. The procedure can easily be scaled up to accommodate larger amounts of cells or tissue.
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4110149A5.qxd 1/15/2003 2:49 PM Page 7 • Reducing agent (for example, DTT, TBP, or TCEP) • Carrier ampholytes (for example, Bio-Lyte 3/10 ampholyte) • RC DC™ Protein Assay (Bio-Rad catalog #500-0121 or #500-0122) Section 3 Storage Conditions The unopened kit can be stored at room temperature. Buffer S1, buffer S2, and PSB diluent should be stored at 2–8°C after opening. Use aseptic technique when handling buffer S1, buffer S2 and PSB diluent to prevent contamination.
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4110149A5.qxd 1/15/2003 2:49 PM Page 8 Section 4 Instructions for Use Note: Chill buffers S1 and S2 on ice for at least 15 min before beginning (invert bottles several times during the incubation). 1. In a microcentrifuge tube, add 0.5 ml of buffer S1 per 25–50 mg of animal tissue or 0.05 ml of wet cell pellet from sources such as cell culture, yeast, or bacteria. For plant tissue add 0.5 ml of buffer S1 per 0.25 g tissue.
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4110149A5.qxd 1/15/2003 2:49 PM Page 9 the sample using 30–40 sec bursts until lysis is complete (typically 3–4 times). Chill the suspension on ice for 1 min between each ultrasonic treatment. Note: Disruption of cells by sonication is dependent on the cell type. For example, E. coli requires longer sonication times than animal cells and tissues.
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4110149A5.qxd 1/15/2003 2:49 PM Page 10 7. The final pellet contains the signal proteins. Resuspend the pellet in 0.2–0.3 ml of complete PSB. Vortex the tube 4 to 5 times for 60 seconds each. Incubate the tube at room temperature for 10–15 min vortexing periodically to ensure the pellet is completely dissolved. Centrifuge the sample at maximum speed in a microcentrifuge for 10 minutes at room temperature and collect the clarified supernatant.
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4110149A5.qxd 1/15/2003 2:49 PM Page 11 Section 5 Appendix 5.1 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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4110149A5.qxd 1/15/2003 2:49 PM Page 13 5.2 Preparation of 2-D Rehydration/Sample Buffers The PSB and PSB diluent are provided with this kit to solubilize the final pellet containing the signal proteins. PSB is a proprietary, strongly chaotropic 2-D rehydration/sample buffer that will solubililze both hydrophilic as well as hydrophobic proteins. Other strongly chaotropic 2-D rehydration/sample buffers (Section 5.2.
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4110149A5.qxd 1/15/2003 2:49 PM Page 14 Component Final Concentration Amount to Make 2 ml DTT * (FW 154.3) 50 mM 15.4 mg 100X Bio-Lyte 3/10 ampholyte** 0.2% (w/v) 20 µl Bromophenol Blue 0.002% (w/v) 4 µl of a 1% (w/v) solution *If TBP or TCEP is substituted for DTT as the reducing agent, use at a concentration of 2 mM. **Use an ampholyte buffer that corresponds to the pH range of the IEF separation to be performed.
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4110149A5.qxd 1/15/2003 2:49 PM Page 15 5.2.2. Strongly Chaotropic 2-D Rehydration/Sample 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 0.002% (w/v) 4 µl of a 1% (w/v) solution 1.
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4110149A5.qxd 1/15/2003 2:49 PM Page 16 Section 6 References 1. Simons K and Ikonen E, Functional rafts in cell membranes, Nature 387, 569–572 (1997). 2. Brown D and Rose J, Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface, Cell 68, 533–544 (1992). 3. Parton RG and Simons K, Digging into caveolae, Science 269, 1398–1399 (1995). 4. Anderson RG et al.
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4110149A5.qxd 1/15/2003 2:49 PM Page 18 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 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.