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10005508A.qxp 8/11/2006 11:50 AM Page C Table of Contents Section 1 Introduction....................................................1 Section 2 Kit Specifications ..........................................1 Section 3 Storage Conditions ........................................3 Section 4 Instructions for Use........................................5 Section 5 Appendix ......................................................9 Section 6 Reference ....................................................
10005508A.qxp 8/11/2006 11:50 AM Page 1 Section 1 Introduction MicroRotofor lysis kits provide convenient, efficient methods for the preparation of protein samples for fractionation with the MicroRotofor cell. The MicroRotofor lysis kit (plant) is designed for use with soft plant tissues and plant cell cultures, and employs tissue maceration and/or solubilization into a chaotropic extraction buffer (Vuillard et al. 1995).
10005508A.qxp 8/11/2006 11:50 AM Page 2 Each MicroRotofor run using 2.5 mg total protein yields ten 150–250 µl fractions, and the protein distribution among the fractions will vary depending on the sample. For example, using spinach leaf extracts and ampholytes spanning the pH range 3–10, fractions 3–5 typically contain the highest amounts of protein. Certificates of analysis and MSDS forms are available upon request.
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10005508A.qxp 8/11/2006 11:50 AM Page 4 Component Protein solubilization buffer (PSB), 25 g PSB diluent, 30 ml ReadyPrep 2-D cleanup kit Precipitation agent 1, 15 ml Precipitation agent 2, 15 ml Wash reagent 2, 50 ml Wash 2 additive, 0.25 ml Wash reagent 1, 2.
10005508A.qxp 8/11/2006 11:50 AM Page 5 Section 4 Instructions for Use Preparation of Protein Solubilization Buffer (PSB) Solution 1. Use only freshly rehydrated buffer. Discard any unused buffer. 2. Allow the PSB dry reagent to warm to room temperature before opening the bottle. Shake the PSB dry reagent bottle for 10–15 sec. Weigh an appropriate amount (each gram of dry reagent will prepare approximately 2 ml buffer solution). Use 1 ml of freshly prepared PSB per 300 mg of plant tissue (Table 1).
10005508A.qxp 5. 8/11/2006 11:50 AM Page 6 Add reducing agents, protease inhibitors and carrier ampholyte as needed (Table 2).. Table 2. Additions to PSB solution recommended for various applications. Note that though the applications listed often require use of chaotropes and detergents, these agents are already included in the PSB solution. Component Protein Extraction IEF Separation MicroRotofor Cell IPG Strip Carrier ampholyte NA 2% (w/v) 0.
10005508A.qxp 8/11/2006 11:50 AM Page 7 8. Resuspend 1 g of powdered sample in 3 ml of prepared PSB. Vortex briefly, then sonicate ten times for 20 sec each in an ice bath. 9. Centrifuge at 20,000 x g for 30 min at 20°C. 10. Remove any floating debris by filtering the supernatant using spin filters. If using Bio-Rad's spin filters, centrifuge at 12–16,000 x g for 10 min. Collect filtrate. 11. Determine the protein concentration of the extract.
005508A.qxp 8/11/2006 11:50 AM Page 8 12. Prepare fresh PSB solution containing PSB diluent, glycerol, carrier ampholyte, and DTT or TBP (DTT or TBP is not required if a reduction-alkylation step is performed at step 11). See Table 2 for recommendations. 13. One MicroRotofor run requires ~2.5 mg protein (1 µg/µl) in a total volume of 2.5 ml. Using the above prepared solution, prepare 2.5 ml of a 1 µg/µl dilution of the protein extract.
10005508A.qxp 8/11/2006 11:50 AM Page 9 For subsequent analysis of MicroRotofor fractions by 2-D PAGE, the ampholyte concentration in samples should not exceed 0.2–0.5%. If fractions contain high amounts of protein, dilution prior to loading onto the IPG strip (by 1:10 or greater) will be sufficient to reduce the ampholyte concentration. In cases where protein levels are lower, use of the ReadyPrep 2-D cleanup kit (catalog #163-2130) for ampholyte removal is recommended.
10005508A.qxp 8/11/2006 11:50 AM Page 10 Table 3. Recommended protein loads for IPG strips. IPG Strip Length 7 cm Rehydration volume/strip 125 µl Protein load Silver stain Coomassie G-250 11 cm 17 cm 18 cm 24 cm 185 µl 300 µl 315 µl 410 µl 5–20 µg 20–50 µg 50–80 µg 50–80 µg 80–150 µg 50–100 µg 100–200 µg 200–400 µg 200–400 µg 400–800 µg 2.5–75 µg 10–150 µg 25–300 µg 25–300 µg 40–600 µg Flamingo™, SYPRO Ruby The suggestions made in Table 3 are a general rule of thumb.
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10005508A.qxp 8/11/2006 11:50 AM Page 12 Buffer Components 161-0611 163-2101 163-2091 163-2094 161-0737 Dithiothreitol (DTT), 5 g Tributylphosphine (TBP), 200 mM, 0.6 ml ReadyPrep Proteomic Grade Water, 500 ml Bio-Lyte® 3/10 Ampholyte, 100x, 1 ml Laemmli Sample Buffer, 1x, 30 ml Coomassie is a trademark of BASF Aktiengessellschaft. SYPRO is a trademark of Molecular Probes Inc.
10005508A.qxp 8/11/2006 11:50 AM Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
