Notes: Delta-like 1 homolog (DLK1) functions in cell differentiation during development in both a Notch-dependent and -independent manner. Here, the CheckMate™/Flexi® Mammalian Two-Hybrid System and Dual-Luciferase® Reporter Assay System are used to monitor DLK1-DLK1, DLK1-fibronectin, and DLK1-cysteine-rich FGF receptor interactions. This further illustrates the function of the Notch-independent mechanism in development. (5104)

Notes: The BRD9 subunit of the SWI-SNF chromatin-remodeling complex is investigated in the context of aberrant cell proliferation in acute myeloid leukemia. Specifically, the NanoBRET system was used to measure the interactions of BRD9 and Histone H3.3. A small molecule inhibitor of the BRB9 bromodomain function, BI-7273, was further assessed in HEK293T cells. Disruption of BRB9 interaction with Histone H3.3 was confirmed at sub-micromolar concentrations of BI-7273. Cell viability in the presence of inhibitor was determined using the CellTiter-Glo System. (5079)

Notes: Cell pellets of A375 human melanoma cells were lysed by passing through a needle, centrifuged to remove debris and protein quantitated. Twenty micrograms of protein from control and treated lysates were digested with 1µg of Sequencing Grade Modified Trypsin and used for mass spectrometry analysis. A375 cells (5 × 10⁵) were grown in a 10cm dish and treated for 24 hours before isolating RNA and DNA synthesized using M-MLV Reverse Transcriptase. The cDNA was then used in qPCR. A375 cells were plated at 3 × 10³ in 96-well half-volume white-wall plates, grown and treated for 24 hours. NAD, NADH and NADPH were determined using the NAD(P)H-Glo™ Detection System or NAD/NADH-Glo™ Assay and luminescence measured. (4963)

Notes: These authors analyzed and compared affinity-purified protein complexes from brain homogenates of wild type and huntingtin (Htt) mutant mice by mass spectrometry. Brain tissue from FLAG-tagged wild-type and Htt mice was homogenized in HEPES buffer supplemented with protease inhibitors and RNasin®. After affinity purification, protein complexes were digested using Sequencing-Grade Modified Trypsin and ProteaseMAX™ Trypsin Enhancer prior to mass-spectrometry analysis. (4227)

Notes: The authors explore barley (Hordeum vulgare) as a means of expressing recombinant human Flt3 ligand, which is a growth factor involved in proliferation and differentiation of stem cells and development of various immune cells. As part of their quality control, they performed in-gel proteolytic digestion and mass spectrometry. The recombinant Flt3 ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Coomassie® blue staining, excision of the protein band, destaining, drying of the gel slice and digestion with Sequencing Grade Modified Trypsin at 30°C overnight prior to mass spectrometry. To test biological activity, the authors treated human acute myeloid leukemia cells with Flt3 expressed in barley or a commercial source of Flt3 then measured cell proliferation using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay. (4352)

Notes: The protein phosphatase Cdc14 is sequestered in the nucleolus during interphase and, after successful interphase, is dispersed from the nucleolus throughout the cell by an unknown mechanism to drive a cell's exit from mitosis. The authors determined that Cdc14 contains a nuclear localization signal (NLS), but phosphorylation of serine and threonine residues adjacent to the NLS interferes with localization. These phosphorylation sites were mapped using trypsin and AspN digestion, followed by mass spectrometry. The authors showed that phosphorylation of Cdc14 is mediated by the protein kinase Dbf2-Mob1 by co-incubating purified Dbf2-Mob1 and Cdc14 in the presence of γ-[³²P]ATP. Dbf2-Mob1 was expressed with a His6 tag and purified using MagneHis™ Ni-Particles. (4120)

Notes: The authors identified the maize homolog of hcf136 (Zmhcf136), a gene involved in photosynthesis, and used an RNA blot to determine if ZmHcf136 transcripts accumulate preferentially in mesophyll cells. DNA probes for Zmhcf136 and several cell-specific markers were generated by PCR using GoTaq^® Green Master Mix, gel purified and radiolabeled prior to use in the RNA blots. To examine differences in protein accumulation and localization in wildtype and hcf136 mutants, proteins from subcellular fractions were subjected to two-dimensional gel electrophoresis, and spots of interest were excised, digested with Sequencing Grade Modified Trypsin, then analyzed by electrospray ionization-tandem mass spectrometry. (3883)

Notes: This article examines the interaction between CAR, a member of the nuclear steroid/thyroid hormone receptor family, which translocates from the cytoplasm to the nucleus when cells are exposed to phenobaritol, and the membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A), which is a novel CAR-binding protein. The R16A protein, it was expressed using the TnT^® Coupled Reticulocyte Lysate System and labeled with ³⁵S. This protein was incubated with GST-hCAR-fusion protein attached to a glutathione resin; the resin was washed, and the bound protein was separated by PAGE and detected by autoradiography. Affinity-tagged R16A was expressed in HepG2 cells, purified and separated by SDS-PAGE. The two major bands were excised, digested with Sequencing Grade Modified Trypsin, lyophilized, resuspended in acetonitrile and subjected to mass spectrometric analyses. The CheckMate™ Mammalian Two-Hybrid System was used to examine the interaction between wildtype and mutated R16A. Forty-eight hours posttransfection into HepG2 cells or 16 hours after DNA injection into mice and liver homogenization, the luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. (3752)

Notes: For protein expression profiling of cancer cells treated with 17-allylamino-17-demethoxygeldanamycin (17AAG), a compound with known antitumor activity, lysates were prepared from A2780 cells, a human ovarian adenocarcinoma cell line, and subjected to two-dimensional protein analysis. The spots that varied in intensity in 17AAG-treated cells when compared to control cells were excised and the protein gel slice was reduced, alkylated and in-gel digested using Sequencing Grade Modified Trypsin. (3601)

Notes: The authors characterized a juvenile hormone response element in Drosophila melanogaster (DmJHRE1) and identified two proteins that bound to a DmJHRE1 affinity column. Proteins eluted from the column were digested with Sequencing Grade Modified Trypsin, subjected to liquid chromatography-tandem mass spectrometry and identified as FKBP39 and Chd64. DmJHRE1 transcription regulatory activity was confirmed using reporter constructs with DmJHRE1 sequences regulating expression of firefly luciferase in Drosophila L57 and S2 cells. A vector with Renilla luciferase and the Autographa californica multicapsid nucleopolyhedrovirus IE1 promoter was used for normalization. Luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. Potential interactions between FKBP39, Chd64 and several candidates proteins for the JH receptor were examined using the MagneGST™ Pull-Down System. Each bait protein was expressed as a GST-fusion protein in E. coli and immobilized using MagneGST™ Glutathione Particles. [³⁵S]Methionine-labeled prey proteins were expressed using the TNT^® T7 Quick Coupled Transcription/Translation System. (3784)

Notes: Native uncoupling protein 1 (UCP 1) was purified from rat mitochondria by hydroxyapatite chromatography, separated by SDS-PAGE and the band stained with Coomassie^® Brilliant Blue G-250. The 30–33kDa UCP 1 band was excised, destained, dehydrated, reduced and alkylated. The protein was then digested overnight with 40µl of 20ng/µl Sequencing Grade Modified Trypsin. The resulting peptides were extracted and analyzed by mass spectrometry. (3331)

Notes: Prohibitin (PHB-1) is a multifunctional protein that is located in the mitochondria and plasma membrane and is secreted by adipocytes. Previously PHB-1 was shown to function as a chaperone protein for newly made subunits of mitochondrial respiratory enzymes. This paper describes a role for exogenous PHB-1 in the modulation of insulin-stimulated glucose and fatty acid oxidation. To identify PHB-1 binding partners, His-tagged PHB-1 was incubated with adipocytes. Membranes were solubilized and proteins separated by electrophoresis. Silver-stained bands were excised and destained before treatment with sequencing grade Trypsin. The tryptic peptides were analyzed by HPLC and Mass Spectrometry. (3634)

Notes: The authors wanted to learn more about the proteins that comprise the Tetrahymena
phagosome proteome. Proteins were isolated from phagosome preparations, denatured by adding 5mM dithiothreitol and heating to 60°C for 1 hour. The samples were cooled to room temperature and alkylated by incubation with 10mM iodoacetamide for 1 hour protected from light. Sequencing Grade Modified Trypsin, at a 1:20 concentration (wt/wt) with an equal volume of 50mM ammonium bicarbonate, was added to the sample and incubated overnight at 37°C. Each sample was analyzed by two-dimensional liquid chromatography–tandem mass spectrometry (LC-MS/MS). Whole-cell Tetrahymena DNA was isolated using the Wizard^® Genomic DNA Purification Kit protocol for isolating genomic DNA from plant tissue omitting the use of liquid nitrogen with mortar and pestle. The isolated DNA was then used for restriction digestion, ligation and sequencing. (3680)

Notes: The authors used tetanus toxin C fragment (TTCF) antigen as a model to show that asparagine deamidation interferes with processing by asparagine endopeptidase (AEP) and can influence antigen presentation in T cells. The level of iso-aspartic acid, a product of Asn deamidation, in aged TTCF was quantitated using the IsoQuant^® Isoaspartate Detection Kit. Asn deamidation was confirmed by tryptic digestion using Sequencing Grade Modified Trypsin, followed by MALDI-TOF mass spectrometry. The observed sizes of two tryptic peptides were one mass unit larger than expected, consistent with the fact that Asn deamidation leads to a gain of one mass unit. (3664)

Notes: To identify proteins expressed on the cell surface of various strains of Leptospira, the authors labeled intact Leptospira cells with biotin, solubilized the cells with Triton^® X-100, then captured the biotinylated proteins using the SoftLink™ Soft Release Avidin Resin. The captured products were analyzed by one- and two-dimensional gel electrophoresis. The spots were excised and washed with 50mM ammonium bicarbonate /100% acetonitrile. The gel pieces were then dried, rehydrated in a solution containing 12ng of Sequencing Grade Modified Trypsin, and incubated in 50mM ammonium bicarbonate overnight at 37°C. The proteins were concentrated, desalted and subjected to MALDI-TOF mass spectrometry to identify the individual proteins of the Leptospira surfaceome. (3661)

Notes: The GG2 element located upstream of the human insulin gene was mutated and cloned into a firefly luciferase construct. Two pancreatic mouse cell lines, ßTC-3 and Min6, were co-transfected with the various GG2 luciferase vectors using phRL-TK Vector as a normalization control. Luciferase expression was then assessed using the Dual-Luciferase^® Reporter Assay System. Three factors known to affect insulin gene expression were transcribed and translated using the TNT^® Coupled Reticulocyte Lysate System. The proteins were then used in a gel-shift assay with several DNA element oligos. A 38-40 kDa protein that bound to the GG2 element was identified.  This protein was isolated by DNA affinity chromatography, run on a SDS-PAGE gel and digested with 0.01µg/µl Sequencing Grade Modified Trypsin. Digestion products were then analyzed by Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and TOF/TOF tandem mass spectrometry. (3116)

Notes: The authors cloned and sequenced the dsRNA from Penicillium chrysogenum virus (PcV) and analyzed the gene products of the segmented virus. After reverse transcription followed by PCR to confirm the 5’ and 3’ ends of the dsRNA segments, RT-PCR was used to amplify the complete cDNAs for the four viral segments. The products were cloned into pGEM®-T Vector after A-tailing. To confirm the ORFs predicted for each of the four segments, the cDNAs were added to TNT® T7 Quick Coupled Transcription/Translation System reactions that included radiolabeled methionine. The resulting proteins ranged in size from 94-128 kDa and were separated on an 8% SDS-PAGE gel and analyzed by autoradiography. Gradient-purified PcV virions were digested with Sequencing Grade Modified Trypsin at 37°C for 18 hours and the digestion products were separated by reverse-phase HPLC on a C18 column. Two highly resolved peptides were selected for amino acid sequencing by automated Edman degradation. (3089)

Notes: Promega’s Sequencing Grade Modified Trypsin was used to perform in-gel digests of Coomassie^® blue-stained proteins that associated with Nek-9 in Nek-9 immunoprecipitates from HeLa cell extracts.  Digestions were performed on dried 7.5 or 10% polyacrylamide gel slices and peptide fragments were analyzed by Electrospray mass spectrometry. The two analyzed proteins were sequenced and determined to be SSRP1 and FACT140.  (3057)

Notes: In this paper, accelerated in-gel digestions were demonstrated with Promega’s Sequencing Grade Modified Trypsin. Tryptic digestion of BSA in dried gel slices was tested.  Other variables such as gel slice surface area and temperature of digestion were also tested with Promega’s Sequencing Grade Modified Trypsin.  Accelerated in-gel tryptic digest peptides from various proteins were analyzed by MALDI-TOF and scored based on peak number and a statistically significant MOWSE score.  The accelerated procedure was demonstrated to be more efficient and accurate than a conventional procedure. (3056)

Notes: For mass spectrometric analyses of yeast Hub-1 protein conjugates, SDS-PAGE-purified immunoprecipitates were digested overnight with modified trypsin. (2591)

Notes: To clone the SUMO coupling enzyme, the SUMO peptide was attached to a column, and HeLa extracts were run through the column. Eluted proteins were separated by SDS-PAGE, stained and the appropriate bands were cut from the gel. The gel bands were then digested with an in-gel trypsinization protocol using the Sequencing-Grade Modified Trypsin. The resultant bands were separated by HPLC, sequenced and the full sequences were obtained from the ATCC database. The SUMO-1 Activating enzyme was found to contain two subunits, SAE-1 (38kDa) and SAE-2 (72kDa). These subunites were expressed in vitro using the TNT^® Coupled Wheat Germ Extract System and found to interact. (1226)

Notes: The Sequencing Grade Modified Trypsin was used to digest a protein isolated from a gel slice. The protein was isolated by a referenced method. The isolated protein was dehydrated and then digested overnight at 37°C in 75µl of 200mM NH₄HCO₃ containing 2.5µg of the modified trypsin. The resulting peptides were recovered by extraction with 60% acetonitrile and 0.1% trifluoroacetic acid and subsequent HPLC to isolate individual fragments for sequencing. (0824)

Notes: Flock House Virus (1mg/ml) was digested with Sequencing Grade Modified Trypsin in a reaction with a 1:3000 enzyme to virus ratio in a 10-20µl volume. At each time point, 0.5µl of the reaction was removed and placed on a MALDI analysis plate. The resulting cleavage products were treated with the exprotease carboxypeptidase YY to obtain c-terminal sequence information. (1389)

Notes: The Sequencing Grade Modified Trypsin was used to digest a protein on a nitrocellulose membrane. Digestion prior to protein sequencing are referenced. (0815)

Notes: Complexes of the protein NTH1 and an oligonucleotide were covalently crosslinked. The complex (12nmol) was resolved by ion-exchange chromatography and dialyzed against 50mM NH₄HCO₃. The complex was boiled for 5 minutes and digested with 20µg each of Sequencing Grade Modified Trypsin and Sequencing Grade Endoproteinase Glu-C (discontinued) at 37°C for 2 hours, and the digest was repeated for an additional 48 hours. The resulting peptides were resolved by HPLC and peptides sequenced. (0991)