Exacting Extractions

"Innovation is our mission," states Rockford, Ill.-based Pierce Chemical Company's Rick Chu. True to those words, Chu has applied Pierce's expertise in detergent chemistry to the development of a family of protein extraction reagents designed to rapidly and efficiently extract proteins from bacteria, yeast, and mammalian cell lines. Cell lysis, of course, is an important first step in recovering recombinant proteins or for providing samples for various assays. The new Pierce protein extraction reagents provide a gentle means of cell lysis that is not detrimental to the protein of interest. The first-born member of this family, the B-PER Bacterial Protein Extraction Reagent, consists of a proprietary nonionic detergent in 20 mM Tris*HCl buffer (pH 7.5). In use, a 10- to 20-fold volume of B-PER™ Reagent is added to a bacterial pellet and shaken for 10 minutes. Soluble proteins are then recovered by a centrifugation step, and inclusion bodies are purified from the pellet to near homogeneity with an optimized procedure. This gentle, nonmechanical method is nondenaturing and produces yields exceeding those obtained by standard sonication methods, yet it is scalable for any size protein extraction, from 1.5 to 250 ml or larger cultures. And if necessary, dialysis will remove the nonionic detergent. B-PER Reagent's success and utility as a protein extraction tool has spawned some offspring. B-PER II Bacterial Protein Extraction Reagent, for example, facilitates the extraction of soluble protein and inclusion bodies from smaller culture volumes (less than 10 ml) while retaining all the features and benefits of the original B-PER Reagent. B-PER Protein Extraction Reagent (in phosphate buffer) is comparable to the original B-PER Reagent except that the Tris buffer has been replaced with a phosphate buffer. With this product, designed for proteins where a phosphate buffer system is preferred, Pierce has demonstrated that the change in buffer has no impact on extraction efficiency. 6xHis- and GST-tagged fusion protein purification has also been simplified with the application of B-PER Reagent to these techniques. Pierce manufactures two kits designed for use when more than 1 mg of fusion protein is required. The first, the B-PER 6xHis Column Purification Kit, enables the purification of 6xHistidine-tagged fusion proteins from bacteria in about 2.5 to 3 hours. After lysis with the B-PER Reagent, the 6xHis-fusion protein is recovered from the lysate by affinity chromatography on a nickel-chelated column. Five columns are included in the kit, and up to 10 mg of protein may be purified per column. The second kit, known as the B-PER GST Column Purification Kit, has been designed for the purification of glutathione S-transferase (GST) fusion protein from bacteria. After extraction from the bacteria with the B-PER™ Reagent, the GST fusion protein is recovered by affinity chromatography on an immobilized glutathione column. Overall, this process requires about 2 hours. To speed up the process, Pierce has created spin column versions of these kits designed for the purification of about 1 mg of each fusion protein in under 30 minutes. Pierce has expanded the product line by applying the successful B-PER technology to the extraction of proteins from yeast and mammalian cells. The Y-PER Yeast Protein Extraction Reagent, for instance, is a mixture of proprietary compounds that lyses yeast cells in 20 minutes. Designed as a hassle-free replacement for mechanical lysis of yeast cells with glass beads, the Y-PER Reagent yields up to twice as much protein as the glass bead method. The product has been tested on Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Bacillus subtilis and successfully extracts functionally active proteins such as ß-galactosidase. M-PER Mammalian Protein Extraction Reagent, the latest product in Pierce's line of protein extraction reagents, incorporates a unique detergent with excellent properties for cultured mammalian cell lysis. Developed as a replacement for freeze-thaw cycles and sonication methods, M-PER Reagent extracts up to 25% more protein from mammalian cells than these standard procedures. In fact, in some cases, M-PER Reagent extracts up to three times more protein than competitive products. M-PER Reagent is suitable for protein extraction from several cultured mammalian cell lines, partly because the hydrophobic and hydrophilic groups on the reagent's proprietary detergent provide the versatility needed to lyse the different membranes found in the various cell types. Additionally, M-PER Reagent successfully lyses cells on all sizes of culture plates without scraping. Because Pierce has demonstrated M-PER Reagent's compatibility with several downstream assays, such as reporter assays, kinase assays, and immunoassays, the reagent's ability to lyse cells directly on plates makes this product highly desirable for high-throughput cell lysis and screening assays. The author, Michael Brush, can be contacted at mdbrush@compuserve.com. For more information, please contact Pierce at (800) 874-3723 or visit the company's Web site at www.piercenet.com.

Exacting Extractions

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