RESUMO
Quantitative protein analysis of single cells is rarely achieved due to technical difficulties of detecting minute amounts of proteins present in one cell. We develop a mix-and-read assay for drop-based label-free protein analysis of single cells. This high-throughput method quantifies absolute, rather than relative, amounts of proteins and does not involve antibody labeling or mass spectrometry.
RESUMO
Ribonucleases (RNases) with different sequence or structural specificities are used for a variety of analytical purposes, including RNA sequencing, mapping, and quantitation. The development of RNase protection assays, structural determination assays, and the production of small interfering RNAs (siRNA) employed in RNA interference (RNAi) experiments has depended on the unique substrate specificities of commercially available RNases, including RNases A, I, T1, V1, HI, III, and Dicer. One very common application for high purity RNase A is also presented in this unit and involves hydrolyzing RNA that contaminates DNA preparations. RNase HII and the placental RNase inhibitor are also discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/metabolismo , Ensaios de Proteção de Nucleases/métodos , Proteínas de Plantas/metabolismo , Ribonucleases/metabolismo , Análise de Sequência de RNA/métodos , Animais , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Humanos , Proteínas de Plantas/antagonistas & inibidores , RNA/química , RNA/genética , RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Ribonucleases/antagonistas & inibidoresRESUMO
Terminal deoxynucleotidyl transferase (TdT), is a template-independent DNA polymerase that catalyzes the incorporation of deoxynucleotides at the 3'-hydroxyl terminus of DNA, accompanied by the release of inorganic phosphate. TdT does not require a template and will not copy one. Reaction conditions and some applications are described in this unit, including cloning DNA fragments, labeling the 3' terminus of DNA with (32)P or nonradioactive tags, synthesizing model polydeoxynucleotide homopolymers, and detecting DNA damage and apoptotic cells.
Assuntos
Clonagem Molecular/métodos , DNA Nucleotidilexotransferase/metabolismo , Marcação In Situ das Extremidades Cortadas/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Nucleotídeos/metabolismo , Moldes GenéticosRESUMO
The leukemia-associated protein EVI1 possesses seven zinc fingers within an N-terminal domain (amino acids 1-250) that binds to GACAAGATA. Single amino acid missense mutants of EVI1 were developed that failed to bind DNA either in vitro, as assessed by gel shift assay, or in vivo, as shown by transactivation studies. Specifically, mutation R205N lacks high affinity binding to the GACAAGATA motif. Putative EVI1 target genes were identified by using an EVI1-(1-250)-VP16 fusion protein that acts as a transcriptional activator with the binding specificity of EVI1. Sixteen genes induced in NIH 3T3 cells by wild type EVI1-VP16 but not by mutant forms were identified. Sequence analysis revealed evolutionarily conserved GACAAGATA-like motifs within 10 kb of their transcription start sites, and by chromatin immunoprecipitation in fibroblasts, we showed occupancy of many of these sites by EVI1-VP16. To assess whether native EVI1 binds to these sites in EVI1-transformed myeloid cells, we performed chromatin immunoprecipitation in 32Dcl3 and NFS58 cells, using anti-EVI1 antisera, and we showed that the majority of these sites is bound by wild type EVI1. These putative target genes include Gadd45g, Gata2, Zfpm2/Fog2, Skil (SnoN), Klf5 (BTEB2), Dcn, and Map3k14 (Nik). In this study we demonstrated for the first time that the N-terminal DNA binding domain of EVI1 has the capacity to bind to endogenous genes. We hypothesized that these genes play a critical role in EVI1-induced transformation.
