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4110148A5.qxd 1/15/2003 2:49 PM Page 2 Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
4110148A5.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………………….............5–8 Section 5 Appendix……………..……………..............9–13 Section 6 References……………..……………...............14 Section 7 Product Information………………….........
110148A5.qxd 1/15/2003 2:49 PM Page 4 Section 1 Introduction The ReadyPrep protein extraction kit (membrane I) is a simple, rapid and reproducible method to prepare cellular protein fractions highly enriched in membrane and hydrophobic proteins. This kit is part of a group of related kits from Bio-Rad developed to reduce sample complexity in order to improve the chances of identifying lowabundance proteins, such as membrane proteins, and to simplify proteomic studies.
4110148A5.qxd 1/15/2003 2:49 PM Page 5 the detergent-rich phase. Cytoplasmic proteins partition into the aqueous phase. An insoluble pellet is also formed and is a source of more complex membrane proteins. Proteins extracted into the two phases are treated using the ReadyPrep 2-D cleanup kit (provided) to remove agents in the extraction process that interfere with IEF.
4110148A5.qxd 1/15/2003 2:49 PM Page 6 Section 2 Kit Specifications Each ReadyPrep protein extraction kit (membrane I) 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.
4110148A5.qxd 1/15/2003 2:49 PM Page 7 • Protease inhibitor(s) • 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 M1, buffer M2, and PSB diluent should be stored at 2–8°C after opening. Use aseptic technique when handling buffer M1, buffer M2, and PSB diluent to prevent contamination.
4110148A5.qxd 1/15/2003 2:49 PM Page 8 Section 4 Instructions for Use 4.1 Membrane Extraction Protocol Note: Chill buffers M1 and M2 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 M1 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 M1 per 0.25 g.
4110148A5.qxd 1/15/2003 2:49 PM Page 9 exercised to prevent heating of the sample. Sonicate 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.
4110148A5.qxd 1/15/2003 2:49 PM Page 10 7. Add 0.5 ml of prechilled buffer M2 (the same volume as used in step 3) to the tube containing the bottom phase. Vortex and incubate as described in step 3. Repeat steps 4 through 6. Again remove the top layer and pool with the top layer that was collected earlier. Label this tube of pooled top layers Hydrophilic Protein Fraction (Refer to the note in step 9 for further instructions on storing this protein fraction). 8.
4110148A5.qxd 1/15/2003 2:49 PM Page 11 4.2 Processing Hydrophilic and Hydrophobic Membrane Protein Fractions for IEF/2-D Analysis 10. Determine the protein concentration of the hydrophilic and hydrophobic membrane protein fractions. The RC DC Protein Assay is recommended. This assay allows accurate protein quantitation in the presence of detergents, reducing agents, and other substances that typically interfere with other protein assays. 11.
4110148A5.qxd 1/15/2003 2:49 PM Page 12 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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4110148A5.qxd 1/15/2003 2:49 PM Page 14 5.2 Preparation of 2-D Rehydration/Sample Buffers The PSB and PSB diluent are provided with this kit 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. Other strongly chaotropic 2-D rehydration/sample buffers (Section 5.2.
4110148A5.qxd 1/15/2003 2:49 PM Page 15 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.
4110148A5.qxd 1/15/2003 2:49 PM Page 16 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.
4110148A5.qxd 1/15/2003 2:49 PM Page 17 Section 6 References 1. Bordier C, Phase separation of integral membrane proteins in Triton X-114, J Biol Chem 256, 1604–1607 (1981). 2. Santoni V et al., Membrane proteomics: Use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoresis properties, Electrophoresis 21, 3329–3344 (2000) .
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4110148A5.qxd 1/15/2003 2:49 PM Page 19 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.
