As recombinant viruses expressing scorpion toxins are moving closer toward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological activity in nontarget organisms. In the past, we purified a large amount of Androctonus australis anti-insect toxin (AaIT) present in the venom of A. australis with an analytical reversed-ph...

We report nucleic acid (NA) adsorption isotherms and elution profiles for silica surfaces and use these to design a miniaturized NA purification unit based on solid-phase extraction with silica beads. The procedure is based on a pressure drop equation for flow through a packed bed and allows estimation of key design parameters such as channel dimensions, liquid flow rates, sample volume, and am...

Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1–3 mg pure protein/g cell pellet. A simpl...

A novel affinity purification method for rapid isolation of vitronectin (VN) from human plasma is described. Recently we have used phage display technology to obtain clones expressing peptides with high binding activity for VN. The isolated "strong VN binders" were covalently coupled to CNBr-activated Sepharose. Human plasma was applied to the column and bound VN was eluted using 0.5 M acetic a...

An alkaline phosphatase (EC 3.1.3.1) of the placental type was isolated from a seminoma type of human testicular cancer tissue and was purified to homogeneity by sulfate-mediated chromatography on a column of Cibacron Blue Sepharose 4B. The purified enzyme had a specific activity of 40.6 kU per gram of protein and was obtained in a yield of 37%. The purification procedure used was simple and ec...

Immunoprecipitation (IP) is one of the most widely used and selective techniques for protein purification. Here, a miniaturised, polymer-supported immunoprecipitation (µIP) method for the on-chip purification of proteins from complex mixtures is described. A 4 µl PDMS column functionalised with covalently bound antibodies was created and all critical aspects of the µIP protocol (antibody immobi...