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Easy BacLysis Protein Extraction Solution
Description:The Easy BacLysis Protein Extraction Solution facilitates the removal of soluble proteins from E. coli by gently disrupting the bacterial cell wall. This precisely formulated product provides a simple, speedy, low-cost alternative to mechanical methods such as French Press or sonication. The proprietary formulation employs a mixture of non-ionic detergents that perforate the cell wall without denaturing soluble protein. Depending on the particular intended application, additional components, such as lysozyme, protease inhibitors, salts, reducing agents, or chelating agents, may be added to the buffer. The buffer may be used for both soluble protein extraction and inclusion body purification from total bacterial cell lysates.
Easy BacLysis Protein Extraction Solution Plus Hercules Endonuclease also gently releases target proteins from E. coli. It also markedly reduces extract viscosity prior to downstream processing. Cells are harvested by centrifugation and then suspended in BacLysis Solution at room temperature. Hercules Endonuclease is then added. During a brief incubation, proteins are released and nucleic acids are digested. Insoluble protein and cell debris are easily removed by centrifugation. The resulting low-viscosity clarified extract is then ready for purification and analysis. The extract is compatible with most chromatography resins. The insoluble fraction can be further processed to yield purified inclusion bodies.
Storage:Store Easy BacLysis Protein Extraction Solution at 4°C. Hercules Endonuclease is supplied in 50% glycerol containing 10 mM Tris-HCl, 20 mM NaCl, and 2 mM MgCl2, pH 8.0. The enzyme is stored at -20°C. DO NOT store at –70°C. Freezing Hercules Endonuclease results in loss of activity.
1.5 ml cultures of E. coli strain BL21 (DE3) containing pET-28a encoding target protein were harvested by centrifugation and resuspended in 300 μl competitor A protein extraction reagent, or Easy BacLysis Protein Extraction Solution. Samples in lysis reagent were treated according to their respective protocols. Extracts were clarified by centrifugation, then supernatants were removed and insoluble fractions were resuspended in 300 μl distilled water. The supernatant and insoluble fractions were analyzed by SDS-PAGE and Coomassie blue staining.