Notes: The authors created amplicons for sequencing using one of the GoTaq® Master Mix products. NGS was performed on a 454 Genome Sequencer FLX. (4919)

Notes: GoTaq® Master Mix was used for PCR of bacterial ribosomal RNA genes prior to pyrosequencing. (4529)

Notes: The authors used a yeast two-hybrid system to identify proteins that interact with the autoimmune regulator (AIRE) protein. DAXX, a multifunctional protein involved in apoptosis and transcription regulation, interacts with AIRE, as shown through coimmunoprecipitation and colocalization studies. Colocalization of AIRE and DAXX in HeLa cells was demonstrated by confocal microscopy using a Monster Green^® Fluorescent Protein-AIRE fusion protein and endogenous DAXX, which was detected using an anti-DAXX primary antibody and an anti-rabbit secondary antibody conjugated with Texas Red fluorophore. However, AIRE and DAXX did not interact in vitro in a GST pull-down assay using a GST-AIRE construct, radiolabeled DAXX protein expressed in a TNT^® system, and MagneGST™ Glutathione Particles, leading the authors to speculate that the interaction is weak or there are scaffold proteins required for protein interaction. To examine the effect of DAXX on AIRE transcriptional activity, the authors transfected COS-1 and HeLa cells with AIRE and DAXX expression constructs and a luciferase reporter plasmid with the human insulin promoter, then performed Dual Luciferase^® Reporter Assays. AIRE induced transcription of the insulin promoter, but coexpression of DAXX suppressed this transcriptional activation. (4153)

Notes: Barcoded adaptors were ligated to cDNA using the Promega LigaFast kit prior to sequencing on an Illumina platform. (4530)

Notes: Sequence variation within a gene, such as single nucleotide polymorphisms (SNPs), can lead to differences in expressions levels of corresponding mRNAs; genes that are self-regulated by this mechanism (cis modulation) are difficult to identify accurately with existing techniques. The authors used bioinformatic and molecular approaches to estimate error rates when identifying cis-modulated transcripts and developed a simple method to detect these transcripts in C57BL/6J F^₁ hybrid mice. This method, which they named allelic specific expression, is RT-PCR-based and uses PCR primers that flank an informative SNP to quantify the differential expression levels of transcripts. GoTaq^® Flexi DNA Polymerase was used in the PCR. (4051)

Notes: The authors examined the methylation status of methylated-in-tumor (MINT) loci and the degree of microsatellite instability (MSI) in colorectal cancer to determine if methylation of MINT loci during the progression of adenoma to cancer was linked to MSI. They used on-slide sodium bisulfite modification and methylation-specific PCR to examine the methylation index in paraffin-embedded tissue blocks containing normal, adenoma and cancer tissues. MSI status was determined using the MSI Analysis System. Patients with instability at more than 4 markers were classified as MSI-high, and patients with instability at more than 1 marker were considered microsatellite-stable. (4106)

Notes: These authors sought to identify protein signatures associated with late-stage human colorectal cancer. They used 2D gel electrophoresis to compare the protein signatures of normal tissue and tumor samples. Proteins of interest were excised from gels, trypsin digested and analysed by reverse phase LC-MS. The data were compared to existing databases to try to identify specific proteins or protein interactions associated with late-stage tumor tissue. ProteaseMax Surfactant was used to enhance trypsin digestion. (4084)

Notes: This study describes an enrichment method for cysteinyl adducts of human serum albumin. Electrospray ionization mass spectrometry was used to detect mercaptalbumin and HSA-Cys34 modifications before and after enrichment. After enrichment, mercaptalbumin was no longer observed in mass spectra. Trypsin and ProteaseMax Surfactant were used to prepare samples for mass spectrometry analysis. (4083)

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: In this paper, FuGENE® HD reagent was used to transiently transfect HEK293 cells. Cells were plated on 24-well plates at 5 x 10e4 cells/well prior to transfection with 0.3µg plasmid DNA.

Notes: The authors identified a bacterial strain that can use the toxin 3-nitropropionic acid (3NPA) as its sole carbon and nitrogen sources and cloned the genes that encode the enzymes responsible for the initial steps in the 3NPA degradation pathway. The Pseudomonas library used to clone these genes was created using genomic DNA isolated using the Wizard^® SV Genomic DNA Purification System. Closely related genes were amplified from other bacterial species for phylogenetic analysis. PCRs were performed using the GoTaq^® Hot Start Polymerase. (4164)

Notes: The authors of this paper demonstrate the utility of the HaloTag^® protein purification system for purifying functional proteins from mammalian cells. To this end five kinases were cloned into HaloTag^® vectors, expressed in and purified from HEK293T cells. To demonstrate functionality of the purified recombinant kinases, activity was measured using the appropriate ADP-Glo™ Assay Kinase Enzyme Systems. (4145)

Notes: The authors examined the heritability of certain eye characteristics that may be associated with glaucoma using a classic twin study with both monozygotic and dizygotic twins. Zygosity of same-sex twins was determined by typing 15 STR loci using the PowerPlex^® 16 System. (4044)

Notes: To better examine the molecular mechanisms of small bowel adenocarcinomas, DNA was extracted from paraffin-embedded tissue and tested for microsatellite instability (MSI) using the MSI Analysis System, Version 1.2. The PCR products were run on the Applied Biosystems 3130 Genetic Analyzer and analyzed using GeneScan® software. If two or more of the BAT-25, BAT-26, NR-21, NR-24 or MONO-27 monomorphic markers had altered lengths, the tumors were designated as MSI unstable. (4112)

Notes: These authors characterized the venom composition of the water snake Cerberus rynchops. As part of the study, whole venom was trypsinized and fractionated by reverse phase HPLC followed by MALDI-MS/MS analysis. For in-solution tryptic digestion, lyophilized crude venom (500 μg) was dissolved in ammonium bicarbonate and precipitated with ice-cold acetone for 3 h at -15 °C. The pellet was resuspended in 1 ml 50 mM ammonium bicarbonate containing 20 μl of 1% ProteaseMAX Surfactant. After reduction with 10 μl 0.5M DTT (at 56 °C for 20 min) and alkylation with 27 μl of 0.55M iodoacetamide (at room temperature for 15 min in the dark), the venom was digested at 37 °C for 3 h using 18 μg trypsin (1 μg/μl) and 10 μl of 1% ProteaseMAX surfactant to improve tryptic cleavage activity. (4081)

Notes: Dysregulation of the NF-kB pathway has been associated with the formation of a wide variety of tumors and other cancers, as well as diseases, including chronic inflammation and immunodeficiency. Because of the association of constitutive NF-kB regulation and tumors, inhibition of the NF-kB pathway by small molecule antagonists was thought to be a means of reversing or halting the growth and spread of tumors. The authors screened compounds from a database (the NCGC Pharmaceutical Collection or NPC) of small molecule compounds: 52% of the compounds have been approved for human or animal use by the FDA, 22% were drugs approved for use in Europe, and another 25% either drugs approved in other countries or compounds that have been tested in clinical trials. The database served as a source from which to rapidly and efficiently identify already approved drugs that inhibited NF-kB. They used a quantitative high-throughput screening format. To identify small molecules that could inhibit the NF-kB pathway, the compounds were initially screened using a cell-based NF-kB lactamase reporter gene assay, with TNFalpha and MG132 as positive controls. (TNFalpha induced NF-kB coupled beta-lactamase activity, while MG132 blocked TNFalpha induction NF-kB-coupled beta-lactamase activity.) After several rounds of screening, 20 compounds were further studied for their NF-kB inhibition, with NF-kB luc2P HEK293 cells. After a concordance rate of 95% between the luciferase and beta-lactamase tests, compounds were additionally examined for their ability to affect caspase 3/7 activity, for the ability to disrupt the electrochemical gradient across the mitochondrial membrane in relation to cellular apoptosis, as well as tests of the inhibitors on cancer cell viability and affects on LDH release, an indicator of cell necrosis.
(4049)

Notes: pGL4.15[luc2P /Hygro] or pGL4.14 [luc2/Hygro] were used. (4265)

Notes: This paper describes a method for detection of polyA RNA in permeablized cells and cell sections. The method is based on incorporation of 5-bromo-2´-deoxyuridine (BrdUTP) into cDNA by AMV reverse transcriptase. The BrdUTP was easily detectable in DNA-RNA duplexes, and undetectable in DNA-DNA duplexes, making it possible to use the method to detect RNA in situ. RNasin® Ribonuclease Inhibitor was used in the reverse transcriptase reaction mix. (4226)

Notes: These authors characterized inflammatory caspase substrates using an enzymatic enrichment method for caspase 1,-4 and -5 cleaved proteins and mass-spectrometry. To confirm that caspase-1 cleaved the putative substrates, some substrates were expressed and fluorescently labeled using the TNT® T7 Transcription/Translation System and the FluorTect™ Green_Lys System. The proteins were then treated with recombinant caspase-1, and the progression of the reaction tracked via SDS-PAGE. The authors also used the CytoTox™ ONE Homogeneous Membrane Integrity Assay to track membrane permeabilization in relation to caspase cleavage and IL-1B release. (4252)

Notes: To help understand the molecular mechanism behind the biogenesis and assembly of photosystem II (PSII), the authors characterized the high chlorophyll fluorescence low psii accumulation19 (lpa19) mutation, which results in defective PSII biogenesis. To examine Lpa19 expression patterns in lpa19 mutant plants, the authors performed Western blot analysis using an anti-Lpa19 antibody raised against purified recombinant His-tagged LPA19 protein. This antigen was purified using the HisLink™ Resin. (4101)

Notes: Certain bacteriophages can promote host cell sporulation under unfavorable conditions to increase survival of the host and prophage. These types of phages, known as spore-converting phages, have been found in terrestrial Bacillus species. In this article the authors examined the effect of prophages on sporulation of 11 Bacillus isolates from the Gulf of Mexico. Potential prophages in the Bacillus isolates were detected by PCR using unique PCR primer sets for each prophage genome and GoTaq® Green Master Mix. One of these isolates, B14905, was examined in more detail; the genome of this isolate was isolated using the Wizard® Genomic DNA Purification Kit, then sequenced. (4091)

Notes: RNA was isolated from plant tissue using the SV Total RNA Isolation System. (4544)

Notes: In this study the luminescence-based BacTiter-Glo System and the GloMax 20/20 luminometer were used to detect ATP concentrations as low as 0.0001 nM in various water samples. The results correlated with those generated using traditional microbial detection methods. (4103)

Notes: To better understand early onset colorectal cancer (CRC), the researchers examined mismatch repair (MMR) deficiency by analyzing microsatellite instability (MSI) of five mononucleotide markers BAT25, BAT26, NR21, NR24, and MONO27 from tumors using the MSI Analysis System, Version 1.2. Instability at ≥3 markers classified the tumors as high MSI, while those unstable at ≤2 markers were designated as microsatellite stable. These results were compared with immunostaining analysis of MMR proteins. (4113)

Notes: 293T cells were seeded in 12-well plates and transfected using 0.4µg of an expression plasmid, 0.6µg of a luciferase reporter plasmid and 60ng of the pRL-SV40 or pRL-TK control vector using FuGENE® 6 transfection reagent. At 24 hours posttransfection, cells were infected with Sendai virus or mouse hepatitis virus, and 8 hours postinfection, luciferase activity was measured using a dual luciferase reporter assay (Promega). (4277)