Notes: The authors developed a high-density protein array using a recombinant peptide library to map the epitope recognized by a commercially available anti-vitamin D receptor (VDR) monoclonal antibody. By screening 2304 overlapping VDR peptides, they were able to identify the 37-amino-acid epitope. The library was created by amplifying the 1.2kb VDR coding region, cleaning the PCR product with the Wizard^® SV Gel and PCR Clean-Up System, sonicating the PCR product, then cloning the VDR fragments into a bacterial expression vector that confers a glutathione-S-transferase (GST) tag. The epitope was verified by showing that the 37-amino-acid sequence was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA); the full-length VDR, also expressed as GST fusion protein, was used as a positive control. These GST fusion proteins were purified using the MagneGST™ Protein Purification System. (4154)

Notes: The MagneGST™ Protein Purification System was used to purify GST fusion proteins of the oncoprotein HPV16 E7 or various mutants of HPV16 E7. The purified GST fusion proteins were used for in vitro binding experiments with steroid receptor coactivator 1 (SRC-1), which was produced using the TNT^® T7 Coupled Wheat Germ Extract System and labeled with the Transcend™ Non-Radioactive Translation Detection System. GST pull-down assays were resolved by Western analysis using streptavidn-horseradish peroxidase and alpha-GST. To determine the effects of endogenously expressed HPV16 E7 on SCR-1-mediated transcription, luciferase reporters under the control of either the IL-8 promoter or an artificial promoter containing three estrogen response elements repeats (3 × EREs) were cotransfected with a Renilla control vector into two human cervical cancer lines (C33A and CaSki) using either the Transfast™ Transfection Reagent or another commercial transfection reagent. The Dual-Luciferase^® Reporter Assay was then used to determine luciferase activity to functionally map the E7-interacting domain and to determine the effects of high- and low-risk PHV E7s on SRC-1-mediated transcription. (3459)

Notes: In this study, researchers examined the role of tumour necrosis factor (TNF) receptor-associated factors (TRAFs) in signaling by the KSHV viral FADD-like interleukin-1-β-converting enzyme (FLICE)/caspase-8-inhibitory protein (vFLIP). The TRAF domain of TRAF2 was expressed in vitro from a plasmid construct using the TNT^® Quick Coupled Transcription/Translation System and labeled with [³⁵S]methionine. vFLIP was cloned in-frame with a carboxy-terminal GST tag, and the recombinant vFLIP–GST fusion protein was expressed in E. coli and purified using the MagneGST™ Protein Purification System. GST protein only was similarly prepared as a control. The vFLIP–GST fusion and control proteins were incubated with the radiolabeled recombinant TRAF2, and protein complexes were collected by GST pull-down, washed thoroughly and subjected to SDS–polyacrylamide gel electrophoresis. In vitro-translated proteins were visualized by autoradiography. (3327)

Notes: To help determine the role of Escherichia coli glgX gene in bacterial glycogen metabolism, researchers cloned the gene into a glutathione-S-transferase expression vector, pDEST15. Expression was induced by 0.2% arabinose for 3 hours and the protein was then purified using the MagneGST™ Protein Purification System. Protein expression was monitored and analyzed by SDS-PAGE and Coomassie blue staining. (3282)