Overview
Centrifuge the vial before adding solvent (10,000 x g for 5 minutes). The lyophilizate may be difficult to visualize. Add solvent directly to the centrifuged vial. Tap the vial to aid in dissolving the lyophilized product. Tilt and gently roll the liquid over the walls of the vial. Avoid vigorous vortexing. Light vortexing for up to 3 seconds is acceptable if needed.
Soluble in pure water to high-micromolar concentrations (5 µM – 1 mM). For long-term storage in solution, it is recommended to prepare a stock solution by dissolving the product in double distilled water (ddH2O) at a concentration between 100-1000x of the final working concentration. Divide the solution into single-use aliquots and store at -20°C. Before use, thaw the relevant vial(s) and dilute to the desired working concentration in your working buffer. It is recommended to prepare fresh solutions in working buffers just before use. Avoid multiple freeze-thaw cycles to maintain biological activity. Avoid exposure to light.
The neurotrophins ("neuro" means nerve and "trophe" means nutrient) are a family of soluble, basic protein growth factors which regulate neuronal development, maintenance, survival and death in the CNS and PNS1.
NGF, the first member of the family to be discovered, was originally purified as a factor supporting and regulating survival, development, function and plasticity of sympathetic and sensory spinal neurons in central and peripheral nervous systems in vivo as well as in vitro2-4. It is synthesized and secreted by sympathetic and sensory target organs and provides trophic support to neurons as they reach their final target5.
Neurotrophin secretion increases in the nervous system following injury. Schwann cells, fibroblasts, and activated mast cells normally synthesize NGF constitutively, however, direct trauma and induction of cytokines combine to increase neurotrophin production in these cells after injury6.
NGF is purified in three forms: the 7S, 2.5S and β, in which the biologically active subunit is the β subunit. The structural hallmark of all the neurotrophins is the characteristic arrangement of the disulfide bridges known as the cysteine knot, which has been found in other growth factors such as Platelet-derived growth factor7.
Additionally, the involvement of NGF was recently discovered in processes such as asthma8, psoriasis9 and wound healing10. The biological effects of NGF are mediated by two receptors: TrkA, which is specific for NGF, and p75NTR, which binds all the neurotrophins11.
For the past three decades, biotinylated derivatives of NGF have been widely used in the literature as useful probes for the study of the binding and the initial intracellular processing of this growth factor by cells bearing TrkA NGF receptor12. First studies using 125I-NGF have visualized the intracellular location of NGF12. A biotinylated derivative of NGF that retains biological activity, in conjunction with the appropriate avidin conjugates and fluorescent or confocal microscopic techniques, improve the sensitivity and resolution in many applications. Some examples of applications are: targeting of liposomes containing biotinylated NGF to cells bearing TrkA NGF receptors13; elucidating the kinetics and route of ligand-induced internalization of the p75 receptor through signaling endosomes in cycling and differentiated PC12 cells using biotinylated derivative of NGF14; electrically controlled NGF-Biotin delivery from biotin-doped conductive polymer15; studying the retrograde axonal transport mechanism of NGF signals from the axon terminal to the cell body16,17.
