A Small-Molecule Inhibitor of Lin28.

New discoveries in RNA biology underscore a need for chemical tools to clarify their roles in pathophysiological mechanisms. In certain cancers, synthesis of the let-7 microRNA tumor suppressor is blocked by an RNA binding protein (RBP) Lin28, which docks onto a conserved sequence in let-7 precursor RNA molecules and prevents their maturation. Thus, the Lin28/let-7 interaction might be an attractive drug target, if not for the well-known difficulty in targeting RNA-protein interactions with drugs.

Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments.

Short interfering RNAs (siRNAs) are mediators of RNA interference (RNAi), a commonly used technique for selective down-regulation of target gene expression. Using an equimolar mixture of A, G, C, and U phosphoramidites during solid-phase synthesis, we introduced degenerate positions into RNA guide and passenger strands so that, when annealed, a large pool of distinct siRNA duplexes with randomized base pairs at defined sites was created. We assessed the randomization efficiency by deep sequencing one of the RNAs.

Blotting from Immobilized pH Gradient Gels: Application to Total Cell Lysates.

Isoelectric focusing as used in the first dimension of two-dimensional gel electrophoresis separates protein isoforms such as those due to phosphorylation and acetylation. The immunoblotting method described here reveals this diversity by a one-dimensional separation. Using commercially available immobilized pH gradient plates or strips, the resolved proteins are transferred to PVDF membranes by diffusion and are probed with protein-specific antibody.

From little helpers to automation.

Western blot technology has continually evolved to enhance sensitivity, speed, and ease of operation. For enhancing awareness to these developments, this brief review outlines a representative selection of methods and devices, many of which are commercial products. In particular, the steps taken towards partial and full automation of western blotting are addressed.

Origins of protein blotting.

The development of protein blotting in its early days is recounted as arising from the need to tackle a specific analytical problem. Combining diverse elements of common methods and simple lab equipment resulted in a procedure of general utility. The expansion of the idea of carrying out immunoassays on membranes as predecessors of microarrays is briefly touched upon.