RESUMO
Many proteins bind to nucleic acids. For the first characterization of novel proteins, a fast and simple technique for testing their nucleic acid binding capabilities is desirable. Here, we describe the use of a northwestern and southwestern blot protocol for the evaluation of the DNA and RNA binding abilities of a novel putative methyl transferase HSPC133 (METTL5).
Assuntos
Southwestern Blotting/métodos , DNA/metabolismo , Proteínas/metabolismo , RNA/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Southwestern Blotting/instrumentação , DNA/química , Sondas de DNA/química , Sondas de DNA/metabolismo , Digoxigenina/química , Humanos , Ligação Proteica , Proteínas/química , RNA/química , Sondas RNA/química , Sondas RNA/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Antígeno SS-BRESUMO
The principle of hybridization analysis is that a single-stranded DNA or RNA molecule of defined sequence (the probe) can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the target), with the stability of the hybrid depending on the extent of base pairing that occurs. Experimentally, the analysis is usually carried out with a probe that has been labeled and target DNA that has been immobilized on a membrane support. Hybridization analysis is sensitive and permits detection of single-copy genes in complex genomes. This unit presents a basic procedure for hybridization analysis with a radiolabeled DNA probe. Despite its lack of embellishment, the protocol gives acceptable results with Southern blots on nitrocellulose and nylon (uncharged and charged) membranes. An Alternate Protocol describes a similar method for probing DNA blots with a radiolabeled RNA probe. A Support Protocol for stripping blots to ready them for reprobing is also provided.
