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| | | YeastBuster™ Protein Extraction Reagent is formulated for a fast, efficient and gentle extraction of soluble active proteins from Saccharomyces cerevisiae and Pichia pastoris cells. The reagent avoids harsh conditions of vigorous mechanical treatment that often result in heat and oxidative degradation of target proteins. The proprietary formulation utilizes a mix of mild detergent, protein stabilization buffer, and tris(hydroxypropyl)phosphine (THP) reducing agent (THP concentrate provided separately). This powerful combination eliminates the inconsistencies associated with tedious mechanical disruption of yeast cells with glass bead abrasives, ultrasonication and pressure disruption, or enzymatic digestion with b-1,3-glucanase lytic enzymes. In addition to greater total protein yields in crude extracts and recovery of enzymatically active protein, the extracts are fully compatible with GST•Bind™ and Ni-NTA His•Bind¨ immobilized metal affinity chromatography (IMAC) purification methods. The reagent is available in 100 and 500 ml sizes.
Features- Gentle, rapid, efficient extraction of proteins from yeast cells
- Eliminates the inconsistencies associated with abrasive grinding, ultrasonication and pressure disruption of yeast cells
- Higher yield of total and enzymatically active soluble proteins as compared with traditional mechanical or other commercially available methods of cell disruption
- Fully compatible with Ni-NTA His•Bind IMAC and GST•Bind affinity purification methods
 Performance comparison of YeastBuster™ Protein Extraction Reagent, another commercial reagent, and the glass bead method
SDS-PAGE analysis (4–20% gradient gel) and Coomassie blue staining of extracted proteins. S. cerevisiae cells containing a recombinant plasmid expressing a 35.6 kDa GST•Tag™/His•Tag¨ fusion protein were grown at 30°C, induced for expression, and harvested at OD600 of 1.2. Cells were collected by centrifugation at 3,000 X g and resuspended in ice cold sterile water. Equal volumes of cells were dispensed into microcentrifuge tubes, and pelleted at 3,000 X g. Cell pellets (~65 mg wet weight) were resuspended in 330 ml of the respective extraction reagents supplemented with 0.5 mM AEBSF and 15 mg/ml benzamidine. The YeastBuster Reagent also included 0.01 volume 100X THP Solution as directed in the protocol. After initial resuspension of pellets by pipetting, YeastBuster and competitor reagent samples were agitated gently at room temperature for 20 min. Glass bead extraction was accomplished by resuspending the 65 mg pellet in lysis buffer containing 50 mM Tris-HCl, 250 mM LiCl, 100 mM (NH4)2SO4, 1 mM DTT, and 2% glycerol, adding approximately 50 ml acid-washed glass beads (100Đ150 mm diameter), and vortexing the sample on high for 4 min with intermittent chilling on ice. All samples were centrifuged at 16,000 X g for 5 min prior to SDS-PAGE analysis. |
Analysis of total protein and reporter activities
Total protein extracted by the three methods was determined using Non-Interfering Protein Assay™ Kit. GST activity was determined using GST•Tag Assay Kit. b-gal activity was determined using the host expressing LacZ. Cells were grown and processed as described above. Samples of the extracts were assayed using Novagen's BetaRed™ b-Galactosidase Assay Kit. Data reflect the average of duplicate assays.
| | YeastBuster | Competitor | Glass Beads |
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| Protein(mg/ml) | 6.1 | 3.2 | 0.65 | | GST(D A340/min) | 0.071 | 0.023 | 0.007 | | b-gal(D A570/min) | 0.113 | 0.003 | 0.187 |
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