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10005510A.qxp 8/11/2006 10:22 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 ....................................................10 Section 6 References ..................................................
10005510A.qxp 8/11/2006 10:22 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 (bacteria) is designed for use with both gram negative and gram positive bacterial cultures, and employs enzymatic digestion of the cell wall (Repaske et al. 1956) followed by solubilization into a chaotropic extraction buffer (Vuillard et al. 1995).
10005510A.qxp 8/11/2006 10:22 AM Page 2 This kit can be used with both gram negative and gram positive bacteria. 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 E. coli extracts and ampholytes spanning the pH range 3–10, fractions 3–6 typically contain the highest amounts of protein. Certificates of analysis and MSDS forms are available upon request.
10005510A.qxp 8/11/2006 10:22 AM Page 3 Items Required But Not Provided • 1.
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10005510A.qxp 8/11/2006 10:22 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 PSB per 50 µl of wet cell pellet (Table 1). Table 1.
10005510A.qxp 8/11/2006 10:22 AM Page 6 3. For each gram of dry reagent, add 1.1 ml of PSB diluent. 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.
10005510A.qxp 8/11/2006 10:22 AM Page 7 Note: For best results, use a wet cell pellet from a freshly grown bacterial culture. Do not use frozen or stored stocks. 7. Gently flick the bottom of the tube to resuspend the cell pellet (no longer than 1 min). If the cell pellet doesn't resuspend readily, the cells may no longer be viable for spheroplast preparation. Use fresh cultures. 8. Mix the lysozyme by gently flicking the vial and add it to the sample (5 µl of lysozyme per 50 µl of wet cell pellet).
005510A.qxp 8/11/2006 10:22 AM Page 8 and/or further lyse some of the spheroplasts. Should you choose to eliminate this step, you may anticipate the migration of lysozyme on a 2-D gel by knowing its pI (9.32) and molecular weight (14.3 kD). 12. Add 500 µl of freshly prepared PSB solution to the spheroplast pellet. 13. Sonicate the suspension with an ultrasonic probe to break down the cell membrane and genomic DNA. Sonication should be performed in an ice bath to prevent heating.
10005510A.qxp 8/11/2006 10:22 AM Page 9 17. Optional: A reduction and alkylation of the sample is recommended at this point in the procedure. Refer to the ReadyPrep reduction-alkylation kit, catalog #163-2090. 18. Store the protein extract at –70°C, apply it directly onto an IPG strip (see Appendix for details), or proceed to step 19. Preparing Extracts for a MicroRotofor Run (See Section 6 of MicroRotofor manual for alternative sample preparation and load conditions.) 19.
10005510A.qxp 8/11/2006 10:22 AM Page 10 Note: Following fractionation with the MicroRotofor, it is recommended to perform an SDS-PAGE analysis profiling all 10 fractions. This will illustrate the protein content of each fraction. See the Appendix for recommendations pertaining to SDS-PAGE analysis of MicroRotofor fractions. For subsequent analysis of MicroRotofor fractions by 2-D PAGE, the ampholyte concentration in samples should not exceed 0.2–0.5%.
10005510A.qxp 8/11/2006 10:22 AM Page 11 Preparation for IEF on an IPG Strip Following step 17, the sample extract can be loaded onto an IPG strip after appropriate dilution. See Table 3 for recommendations on how much protein sample to load onto an IPG strip. Dilution of the sample can be done using protein solubilization buffer (PSB) as a rehydration/sample buffer. However, some critical components need to be added to the PSB solution to make it IPG-compatible (Table 2).
10005510A.qxp 8/11/2006 10:22 AM Page 12 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) Repaske R et al., Lysis of gram-negative bacteria by lysozyme, Biochem Biophys Acta 22, 189–191(1956) Vuillard L et al.
10005510A.qxp 8/11/2006 170-2836 10:22 AM Page 13 MicroRotofor Syringes, 3 ml and 10 ml, 3 each Protein Quantitation Kits (see also bulletin 2610) 500-0121 500-0122 RC DC Protein Assay Kit I, 500 standard assays, bovine γ-globulin standard RC DC Protein Assay Kit II, 500 standard assays, bovine serum albumin standard Buffer Components 161-0611 163-2101 163-2091 163-2094 161-0737 Dithiothreitol (DTT), 5 g Tributylphosphine (TBP), 200 mM, 0.
10005510A.qxp 8/11/2006 10:22 AM Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
