Western Blot Processing Optimization: The Perfect Blot.

Western blot processing is a well-established procedure that includes protein extraction from tissues and cells, gel electrophoresis separation, transfer to a membrane, and immunodetection with specific antibodies. Here, we show that optimization of washing helps to maximize the specific interactions of antigens and antibodies. Performing all washing steps at 4 °C ensures a maximal signal to noise ratio and reduces nonspecific signals.

Comparative structural analysis of TonB-dependent outer membrane transporters: implications for the transport cycle.

TonB-dependent outer membrane transporters (TBDTs) transport organometallic substrates across the outer membranes of Gram-negative bacteria. Currently, structures of four different TBDTs have been determined by X-ray crystallography. TBDT structures consist of a 22-stranded beta-barrel enclosing a hatch domain.

The Escherichia coli outer membrane cobalamin transporter BtuB: structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation.

Gram-negative bacteria possess specialized active transport systems that function to transport organometallic cofactors or carriers, such as cobalamins, siderophores, and porphyrins, across their outer membranes. The primary components of each transport system are an outer membrane transporter and the energy-coupling protein TonB. In Escherichiacoli, the TonB-dependent outer membrane transporter BtuB carries out active transport of cobalamin (Cbl) substrates across its outer membrane.

Substrate-induced transmembrane signaling in the cobalamin transporter BtuB.

The outer membranes of Gram-negative bacteria possess transport proteins essential for uptake of scarce nutrients. In TonB-dependent transporters, a conserved sequence of seven residues, the Ton box, faces the periplasm and interacts with the inner membrane TonB protein to energize an active transport cycle. A critical mechanistic step is the structural change in the Ton box of the transporter upon substrate binding; this essential transmembrane signaling event increases the affinity of the transporter for TonB and enables active transport to proceed.

Crystallization and initial X-ray diffraction of BtuB, the integral membrane cobalamin transporter of Escherichia coli.

BtuB, the cobalamin transporter from Escherichia coli, has been overexpressed, purified and crystallized. The purified protein was solubilized in n-octyl tetraoxyethylene (C(8)E(4)) and was crystallized using sitting-drop vapor diffusion with PEG 3350 and magnesium acetate as precipitants (pH 6.5).