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Examines the conformational stability of a cold-active alkaline phosphatase (AP) from Atlantic cod. Stability of the AP in terms of subunit dissociation and unfolding of monomers; Role of dimer formation in the overall stability of the cod enzyme; Reduction of affinity for inorganic phosphate.

We describe a system for horizontal 1D or 2D PAGE comprising an apparatus and microgels. There is no buffer outside the gel, making handling and sample loading easy. Specially designed electrodes on all four sides allow 2D electrophoresis without gel rotation. Electrophoresis is completed within 20 min and sensitivity is in the subnanogram range. The system is temperature controlled for speed, denaturation of nucleic acid molecules and maintaining molecules single-stranded. The system allows characterization of structure, conformation and damage in complex nucleic acid preparations. Besides quick 1D PAGE, 2D applications include characterization of efficiency of complex molecular procedures, checking quality of biosamples and detecting DNA damage in cells and body fluids. The system should also run protein gels.

Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acids in complex samples according to strandness, conformation and length. Under the non-denaturing conditions of the first electrophoretic step, single-stranded DNA, double-stranded DNA and RNA.DNA hybrids of similar length migrate at different rates. The second electrophoretic step is performed under denaturing conditions (7 mol l(-1) urea, 55 degrees C) so that all the molecules are single-stranded and separate according to length only. 2D-SDE is useful for revealing important characteristics of complex nucleic acid samples in manipulations such as amplification, renaturation, cDNA synthesis and microarray hybridization. It can also be used to identify mispaired, nicked or damaged fragments in double-stranded DNA. The protocol takes approximately 2 h and requires only basic skills, equipment and reagents.