PopCulture® Reagent* is a detergent-based concentrate that can be added directly to cultures of E. coli to effectively extract proteins without the need for cell harvest. Recombinant proteins can be directly screened in the crude extract, or purified by adding an affinity matrix, washing the matrix-target protein complex to remove spent culture medium and cellular contaminants, and eluting the purified protein from the matrix. The entire culturing, extraction, and purification process can be performed in the original culture tube or multiwell plate. This "in-media" protein screening or purification procedure can be adapted to high-throughput roboticprocessing of samples for proteomics research and any application that would benefit from the increased speed and convenience it provides. Successful purification of intact fusion proteins from total culture extracts has been demonstrated using His•Bind® and GST•Bind™ Resins (Grabski 2001). The use of His•Mag™ or GST•Mag™ Agarose Beads enables the entire procedure to be carried out in a single tube without the need for columns or centrifugation. Addition of rLysozyme™ Solution or the use of pLysS hosts increases the efficiency of protein extraction with the procedure. Benzonase® Nuclease may also be added to reduce the viscosity of the extract.
PopCulture Reagent is supplied as a ready-to-use Tris-buffered liquid concentrate that is stable at room temperature.
 
PopCulture Purification Kits
PopCulture Reagent is available bundled with His•Mag and GST•Mag Agarose Beads and corresponding buffers, plus rLysozyme Solution, for convenient extraction and affinity purification using magnetic separation. These kits enable processing of 40 × 3-ml cultures with yields up to 375 µg His•Tag® or up to 150 µg GST•Tag™ fusion protein per 3 ml culture, based on bead binding capacity. For 96-well processing using PopCulture, use RoboPop™ Purification Kits.
* patent pending |
PopCulture Reagent* is a buffered mixture of concentrated detergents formulated to extract proteins from E. coli cells directly in their culture medium. Using this method, cell culture, protein extraction and purification performed in the original culture tube or multiwell plate. PopCulture perforates the E. coli cell wall without denaturing soluble protein and protects protein from the pH extremes produced in high density culture media. Recombinant proteins can be assayed directly or purified by adding an affinity matrix, washing the matrix:target protein complex to remove culture medium and cellular contaminants and eluting the purified protein from the matrix. Purification of fusion proteins from total culture extracts has been demonstrated using both IMAC and GST affinity chromatography methods (1). To further enhance the PopCulture purification procedure, lysozyme and/or Benzonase® Nuclease may be added. Lysozyme cleaves a peptidoglycan bond in the E. coli cell wall, enhancing cell lysis and increasing the yield of protein (1, 2). Proteins may be expressed in a host encoding T7 lysozyme (pLysS host) or exogenous lysozyme may be added after the PopCulture Reagent. Benzonase Nuclease may also be added to degrade endogenous nucleic acids that may interfere with purification due to high viscosity. The PopCulture protein purification procedure is ideally suited to high throughput (HT) robotic processing of samples for proteomics research or any application that would benefit from the increased speed and convenience. The magnetic agarose capture resins (e.g. GST•Bind™ Magnetic Agarose Beads, His•Bind® Magnetic Agarose Beads) are optimal for HT applications since the entire procedure can be performed in a single tube without the need for columns or centrifugation. *patent pending References:
1. Grabski, A., Drott, D., Handley, M., Mehler, M. and Novy, R. (2001). inNovations 13, 1–4.
2. Inouye, M., Arnheim, N. and Sternglanz, T. (1973). J. Biol. Chem. 248, 7247. |
