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10005507A.qxp 8/11/2006 2:33 PM 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 ....................................................10 Section 6 References ..................................................
10005507A.qxp 8/11/2006 2:33 PM Page 1 Section 1 Introduction MicroRotofor lysis kits provide convenient, effective methods for the preparation of protein samples for fractionation with the MicroRotofor cell. The MicroRotofor lysis kit (mammal) is designed for use with mammlian tissues and cell cultures, and employs tissue maceration and/or solubilization into a chaotropic extraction buffer (Vuillard et al. 1995).
10005507A.qxp 8/11/2006 2:33 PM Page 2 cell). Expected protein recovery using this kit is approximately 20% (based on studies using 100 mg rat brain tissue, mouse liver tissue, and Jurkat cells), and the extraction process requires approximately 1 hr. 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 protein sample.
10005507A.qxp 8/11/2006 2:33 PM Page 3 Items Required But Not Provided • 1.
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10005507A.qxp 8/11/2006 2:33 PM 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 PSB per 100 mg of tissue or 50 µl of wet cell pellet (Table 1).
10005507A.qxp 8/11/2006 2:33 PM Page 6 4. Vortex periodically and incubate at room temperature until you have a clear solution (2–3 min). 5. 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.
10005507A.qxp 8/11/2006 2:33 PM Page 7 Sample Processing** 6. If using ReadyPrep Mini grinders, centrifuge the tubes containing resin in a microcentrifuge at 20,000 x g for 20 sec and remove the collected liquid. 7. Weigh out tissue sample (up to 100 mg per ReadyPrep mini grinder), and add it to the tube containing resin. 8. Add 500 µl of prepared PSB solution to the tube containing resin. 9. Grind the sample using the supplied matching pestle.
005507A.qxp 8/11/2006 2:33 PM Page 8 11. Transfer the supernatant carefully into a new tube without disturbing the pellet. 12. Resuspend the residual cell pellet in 250 µl of prepared PSB, and repeat steps 9 and 10. Collect the supernatant and pool with the first supernatant. 13. Determine the protein concentration of the extract. This is best done using the RC DC protein assay (catalog #500-0121 or 500-0122), which is compatible with the detergents and reducing agents present in PSB.
10005507A.qxp 8/11/2006 2:33 PM Page 9 15. One MicroRotofor 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. These recommendations are based on rat brain as a sample source and may vary depending on sample type. Load the entire 2.5 ml sample into the MicroRotofor chamber. It may be necessary to add extra PSB solution to fill the chamber completely, eliminating any void volumes. 16.
10005507A.qxp 8/11/2006 2:33 PM Page 10 Section 5 Appendix Preparation for SDS-PAGE CHAPS, a component of the PSB diluent, may interfere with SDS-PAGE. Remove CHAPS from the extracts (for example, with the ReadyPrep 2-D cleanup kit), or dilute the extracts 1:1 with 1x Laemmli buffer prior to SDS-PAGE. Preparation for IEF on an IPG Strip Following step 13, the sample extract can be loaded directly onto an IPG strip after appropriate dilution.
10005507A.qxp 8/11/2006 2:33 PM Page 11 Table 3. Recommended protein loads for IPG strips IPG Strip Length 7 cm Rehydration volume/strip 125 µl 11 cm 17 cm 18 cm 24 cm 185 µl 300 µl 315 µl 410 µl Protein load Silver stain 5–20 µg 20–50 µg 50–80 µg 50–80 µg 80–150 µg Coomassie G-250 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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10005507A.qxp 8/11/2006 2:33 PM Page 13 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.
10005507A.qxp 8/11/2006 2:33 PM Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
