0005509A.
10005509A.qxp 8/11/2006 11:30 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........................................4 Section 5 Appendix ......................................................9 Section 6 References ..................................................
10005509A.qxp 8/11/2006 11:30 AM 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 (yeast) is designed for use with yeast cultures, and employs enzymatic digestion of the cell wall (Scott et al. 1980) followed by solubilization into a chaotropic extraction buffer (Vuillard et al. 1995).
10005509A.qxp 8/11/2006 11:30 AM Page 2 the fractions will vary depending on the sample. For example, using Saccharomyces cerevisiae and ampholytes spanning the pH range 3–10, fractions 2–4 typically contain the most protein. Certificates of analysis and MSDS forms are available upon request.
10005509A.qxp 8/11/2006 11:30 AM Page 3 • ReadyPrep™ proteomic grade water (catalog #163-2091) or other ultrapure water • β-mercaptoethanol Items Recommended But Not Required • Protease inhibitor (for example, Sigma catalog #P8215) • ReadyPrep reduction-alkylation kit (catalog #163-2090) • ReadyPrep 2-D cleanup kit (catalog #163-2130) Section 3 Storage Conditions Shipped at ambient temperature. Store kit components as individually marked.
10005509A.qxp 8/11/2006 11:30 AM Page 4 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 60 µl of wet cell pellet (Table 1). Table 1.
10005509A.qxp 5. 8/11/2006 11:30 AM Page 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. Component Protein Extraction IEF Separation MicroRotofor Cell IPG Strip Carrier ampholyte NA 2% (w/v) 0.
10005509A.qxp 8/11/2006 11:30 AM Page 6 7. Add 1 µl of β-mercaptoethanol per 100 µl of yeast suspension buffer. 8. Vortex until the cell suspension becomes homogenous. 9. Flick the vial of lyticase enzyme to mix. Add 10 µl of lyticase enzyme per 100 µl of yeast cell pellet. Gently mix. Lyticase will hydrolyze poly-(beta-1,3-glucose) for lysis of the yeast cell wall. 10. Incubate the cell suspension at 37°C for 30–60 min. 11. Centrifuge the suspension at 10,000 x g for 5 min.
10005509A.qxp 8/11/2006 11:30 AM Page 7 13. Add 600 µl of freshly prepared PSB solution to the spheroplast pellet. 14. Sonicate the suspension to break down the cell membrane and the genomic DNA. Sonication should be performed in an ice bath to prevent heating. Sonication should be performed with bursts of 20–30 sec, with chilling of the suspension on ice between bursts. 15. Centrifuge at 20,000 x g for 30 min at 20°C and collect the clear lysate. 16.
005509A.qxp 8/11/2006 11:30 AM Page 8 Preparing Extracts for a MicroRotofor Run (See Section 6 of MicroRotofor manual for alternative sample preparation and load conditions.) 18. 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 17). See Table 2 for recommendations. 19. One MicroRotofor run requires ~2.5 mg protein (1 µg/µl) in a total volume of 2.5 ml.
10005509A.qxp 8/11/2006 11:30 AM Page 9 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. 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.
10005509A.qxp 8/11/2006 11:30 AM Page 10 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 Coomassie G-250 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.
10005509A.qxp 8/11/2006 11:30 AM Page 11 Store at –20°C when frequently used. Store below –70°C for infrequent usage (less than once a month). Lyticase is stable for 1 year at –20°C and for many years below –70°C. Section 6 References Harbers, A et al., Fractionation by liquid-phase isoelectric focusing in the MicroRotofor cell: improved detection of low-abundance proteins, Bio-Rad bulletin 5344 (2005) Scott J et al.
10005509A.
10005509A.qxp 8/11/2006 11:30 AM 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.
10005509A.qxp 8/11/2006 11:30 AM Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
