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4110157A.qxd 6/16/2003 2:21 PM Page 3 Table of Contents Section 1 Introduction…………………………………...1–2 Section 2 Kit Specifications…………………………......3–5 Section 3 Storage Conditions………………………..........5 Section 4 Reagent Preparation…………………...........6–7 Section 5 Instructions for Use…………………...........8–13 Section 6 Appendix……………..…………….................14 Section 7 References……………..……………...............15 Section 8 Product Information………………............
4110157A.qxd 6/16/2003 2:21 PM Page 4 Section 1 Introduction The ReadyPrep protein extraction kit (membrane II) is designed as a simple, rapid and reproducible method to isolate cellular membranes and then to extract the associated integral membrane proteins into a solution compatible with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE).
110157A.qxd 6/16/2003 2:21 PM Page 5 can be applied to a wide variety of biological samples, from animal cells and tissues to yeast, bacteria and plant tissues. After the cells or tissue are disrupted using an ultrasonic probe, the extract is incubated in a Membrane Protein Concentrating Reagent. This high pH buffer strips away proteins weakly-associated with membranes while leaving membrane fragments with their associated integral proteins intact.
4110157A.qxd 6/16/2003 2:21 PM Page 6 Section 2 Kit Specifications Each ReadyPrep protein extraction kit (membrane II) provides sufficient reagents to perform 10 extractions each starting with 100–200 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 less than 3 hr. Items Supplied With Kit • 2-D Rehydration/Sample Buffer 1. 2 vials. Lyophilized.
4110157A.qxd 6/16/2003 2:21 PM Page 7 Items Required But Not Provided • 2.0 ml microcentrifuge tubes (for example, VWR catalog #20170-237). • Disposable round-bottom tubes for plant sample extraction (for example, VWR catalog # 60818-725). • 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 with minimal horizontal streaking.
4110157A.qxd 6/16/2003 2:21 PM Page 8 • ReadyPrep proteomic grade water (Bio-Rad catalog #163-2091) • RC DC™ Protein Assay (Bio-Rad catalog #500-0121 or #500-0122) Section 3 Storage Conditions Store the unopened kit at room temperature. After opening, unused reconstituted ReadyPrep 2-D Rehydration/Sample Buffer 1 should be aliquoted in 1 to 2 ml volumes and stored frozen at –80°C.
4110157A.qxd 6/16/2003 2:21 PM Page 9 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.
4110157A.qxd 6/16/2003 2:21 PM Page 10 Lysis Buffer. Empty the entire contents of one pouch of Lysis Buffer into a 50 ml disposable tube. Add 50 ml of ReadyPrep proteomic grade water or similar quality water to the tube and mix until the solids are completely dissolved. Rinse the pouch with a small portion of this buffer, and return the solution to the 50 ml tube. If desired, protease inhibitors can be added to a portion of this buffer just before use. Membrane Protein Concentrating Reagent.
4110157A.qxd 6/16/2003 2:21 PM Page 11 Section 5 Instructions for Use Extraction of 100–200 mg of sample. 1. Immediately before performing an extraction, prepare Lysis Buffer, Membrane Protein Concentrating Reagent and ReadyPrep 2-D rehydration/sample buffer 1 per the instructions in Section 4. For a standard extraction you will need approximately 15 ml of Lysis Buffer, 60 ml of Membrane Protein Concentrating Reagent and 1 ml of 2-D Rehydration/Sample Buffer 1.
4110157A.qxd 6/16/2003 2:21 PM Page 12 Plant tissue should be ground to a fine powder using a mortar and pestle in liquid nitrogen before addition of Lysis Buffer. 3. With the sample on ice, sonicate the suspension with an ultrasonic probe to disrupt the cells and fragment the genomic DNA. Sonicate the sample using 30 sec bursts, typically 3–4 times, or until lysis is complete. Chill the suspension on ice briefly between each ultrasonic treatment.
4110157A.qxd 6/16/2003 2:21 PM Page 13 5. Remove and dilute the supernatant directly into a beaker or bottle containing 60 ml of the ice-cold Membrane Protein Concentrating Reagent. Stir the suspension slowly on ice for 60 min. 6. Transfer the sample to ultracentrifuge tubes and centrifuge at 100,000 x g for 60 min at 4°C to pellet the membranes and membrane proteins. 7. Remove the tubes from the rotor and mark the position of the pellet. Carefully decant and discard the supernatant. 8.
4110157A.qxd 6/16/2003 2:21 PM Page 14 11. Before performing the extraction of the pellet, complete the preparation of the 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 solution is prepared by adding 10 µl of ReadyPrep TBP reducing agent and the appropriate ampholyte to 0.2 % (w/v) to every 1 ml of reconstituted 2-D Rehydration/Sample Buffer 1.
4110157A.qxd 6/16/2003 2:21 PM Page 15 14. Centrifuge the tube at maximum speed in a microcentrifuge (~16,000 x g) for 10–20 min at 18–20°C to pellet any remaining cell debris. 15. Remove and transfer the supernatant (Membrane Protein fraction) to a clean tube and discard any residual pellet. 16. Determine the protein concentration of the membrane protein sample. The Bio-Rad RC DC Protein Assay (catalog #500-0121 or #500-0122) is recommended for this measurement.
4110157A.qxd 6/16/2003 2:21 PM Page 16 Additional ReadyPrep 2-D rehydration/sample buffer 1 is available and sold separately for this purpose (see Section 8). Note: For some applications (for example using basic pH range IPG strips), the membrane protein sample may require reduction and alkylation treatment before IEF in order to achieve optimal 2-D separation. The ReadyPrep Reduction-Alkylation Kit can be purchased separately for this purpose (catalog #163-2090). 18.
4110157A.qxd 6/16/2003 2:21 PM Page 17 Section 6 Appendix 2-D Rehydration/Sample Buffer 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.
4110157A.qxd 6/16/2003 2:21 PM Page 18 Section 7 References 1. Fujiki Y et al., Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J. Cell Biology 93, 97–102 (1982). 2. Molloy MP et al., Proteomic analysis of the Eschericia coli outer membrane. Eur. J. Biochem. 267, 2871–2881 (2000). 3. Herbert BR et al., Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent. Electrophoresis.
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4110157A.qxd 6/16/2003 2:21 PM Page 20 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.
4110157A.qxd 6/16/2003 2:21 PM Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
