Notes: This paper describes an improved method for on-bead conjugation of antibodies that overcomes the limitations of solution-based protocols (requirement for purified, high-concentration antibodies and the need for multiple buffer changes). The method uses HaloTag technology to immobilize protein A and G onto high-capacity magnetic beads. Antibodies are then captured and labeled on-bead. The authors demonstrate that the method is compatible with amine- and thiol-based chemistries as well as with  mouse and human antibody isotypes, both purified and in cell media. Protein G and Protein A-HaloTag fusion proteins were constructed by cloning the coding sequences for the Fc-binding domains between N-terminal HQ and C-terminal HaloTag sequences. To create Protein A and G beads, the purified Protein G-HaloTag and Protein A-HaloTag constructs were covalently attached to Magne HaloTag Beads--magnetic cellulose beads activated with HaloTag ligands. (4586)

Notes: These authors compared 6 different detection strategies for protein-protein interactions on protein arrays. They expressed HaloTag^® labeled bait proteins in a cell-free expression system, and captured these bait proteins onto coated glass slides using the HaloLink™ Array System. They then compared detection strategies using prey proteins labeled as follows: 1)³⁵S methionine, 2) fluorescence (BODIPY-FL) and 3) biotin labeling of lysine residues using modified Lys tRNA, 4) chemical labeling after expression, 5) HaloTag^® fusion, and 6) N-terminal FLAG tag. The authors evaluated signal:background ratios, adaptability to high-throughput screening, and ease of use. (3999)

Notes: These authors used the Flexi^® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag^® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi^® System and Gateway^® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag^® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

Notes: These authors tested the effect of different washing, drying and storage conditions on the stability of various protein:protein interaction arrays. They tested five different interacting protein pairs and three enzymes, monitoring stability and activity under various processing conditions. They found that addition of 5% glycerol to the wash buffer helped retain enzyme activity during washing and drying. There was significant loss of enzyme activity when slides were stored dry at 4^oC but slides stored at -20^oC in 50% glycerol retained enzyme activity. HaloTag-fused enzymes in cell-free extracts could undergo multiple freeze-thaw cycles without significant effect on enzyme activity, indicating that such extracts could be frozen at -70^oC and used to print small batches of arrays at various intervals over a period of weeks. (3890)

Notes: The Kazusa cDNA Project is constructing a library of more than 1,000 "full ORF" (F-ORF) clones in the Flexi^® Vector System to characterize the function of proteins that are larger than 50kD. (3393)