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4110159A.qxd 6/25/2003 4:07 PM Page 3 Table of Contents Section 1 Introduction………………………………..........1 Section 2 Kit Specifications…………………………......2–3 Section 3 Storage Conditions………………………..........3 Section 4 Reagent Preparation…………………...............4 Section 5 Instructions for Use………………….............5–8 Section 6 Appendix……………..……………...................9 Section 7 Product Information………………….........
4110159A.qxd 6/25/2003 4:07 PM Page 4 Section 1 Introduction The ReadyPrep protein extraction kit (Total Protein) is designed as a simple, rapid and reproducible method to prepare total cellular protein extracts suitable for 2-D gel analysis. The strongly chaotropic extraction solution used in the kit contains the zwitterionic detergent ASB-14, making this solution one of the most powerful solubilizing reagents available for 2-D electrophoresis.
110159A.qxd 6/25/2003 4:07 PM Page 5 Section 2 Kit Specifications Each ReadyPrep protein extraction kit (Total Protein) provides sufficient reagents to perform up to 20 extractions each starting with 50–100 mg of cells or tissue. The procedure can easily be scaled up to accommodate larger amounts of cells or tissue. The entire procedure can be completed in about 45 min. Items Supplied With Kit • 2-D Rehydration/Sample Buffer 1. Two vials. Lyophilized.
4110159A.qxd 6/25/2003 4:07 PM Page 6 • ReadyPrep 2-D cleanup kit (Bio-Rad catalog #163-2130) for plant sample extraction. When working with plant leaf tissue, removal of interfering compounds, such as phenolics, is essential for high quality 2-D gels that are free of horizontal streaks. • Microcentrifuge capable of spinning at 12–16,000 x g while maintaining a temperature between 18–20°C.
4110159A.qxd 6/25/2003 4:07 PM Page 7 Section 4 Reagent Preparation ReadyPrep 2-D Rehydration/Sample Buffer 1:: Add 5.6 ml of ReadyPrep proteomic grade water or similar quality water to one bottle. Swirl the vial gently until the contents are completely dissolved. The solution can be warmed slightly in the palm of the hand or in a water bath to speed the dissolution process. DO NOT heat the solution above 25 to 30°C to avoid the formation of cyanates.
4110159A.qxd 6/25/2003 4:07 PM Page 8 Section 5 Instructions for Use Extraction of 50–100 mg of sample. 1. Immediately before performing an extraction, complete the preparation of 2-D Rehydration/Sample Buffer 1. Prepare only enough complete 2-D rehydration/sample buffer 1 for the number of extractions being performed. Each extraction will require 1.0 ml of the solution. The complete buffer is prepared by adding 10 µl of ReadyPrep TBP reducing agent and the appropriate ampholyte to 0.
4110159A.qxd 2. 6/25/2003 4:07 PM Page 9 In a 2.0 ml microcentrifuge tube add 1 ml of complete 2-D Rehydration/Sample Buffer 1 (see Step 1) per 50–100 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 2–3 ml of 2-D Rehydration/ Sample Buffer 1 per each gram of tissue in, for example, a disposable 14 ml round-bottom tube.
4110159A.qxd 6/25/2003 4:07 PM Page 10 Note: If using sonication, care must be exercised to prevent heating of the sample. The temperature of the sample should not be allowed to rise above 30°C. Similarly, if the sample becomes too cold, precipitation of the urea and thiourea can occur. If this happens, gently warm the sample until the precipitate dissolves before proceeding further. Note: Disruption of cells by sonication is dependent on the cell type. For example, E.
4110159A.qxd 6/25/2003 4:07 PM Page 11 7. Determine the protein concentration of the sample. The Bio-Rad RC DC Protein Assay (catalog #5000121 or #500-0122) is recommended for this measurement. This assay allows for accurate protein quantitation in the presence of detergents, reducing agents, and other substances that typically interfere with other protein assays. 8.
4110159A.qxd 6/25/2003 4:07 PM Page 12 Section 6 Appendix 2-D Rehydration/Sample Buffer 1 Volume Before IEF and 2D gel electrophoresis, the protein sample may need to be diluted to achieve the desired protein load for the chosen stain. To best determine the volume of diluent to use, consider the IPG strip length, the pH gradient of the IPG strip, and the staining or detection method.
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4110159A.qxd 6/25/2003 4:07 PM Page 14 Rehydration/Sample Buffers 163-2083 ReadyPrep 2-D Rehydration/Sample Buffer 1, 10 ml, containing 7 M urea, 2M thiourea, 1% ASB-14, 40 mM Tris base, and 0.
4110159A.qxd 6/25/2003 4:07 PM Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.