This Mouse Neuroligin 1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Neuroligin 1 protein (Cat: 50725-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the mouse NLGN1 isoform 1 (Q99K10-1) extracellular domain (Met 1-Ser 697) was expressed, with a C-terminal polyhistidine tag.
The recombinant mouse NLGN1 consists of 671 amino acids and predicts a molecular mass of 73.5 KDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rmNLGN1 is approximately 100-110 KDa as a result of glycosylation.
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Neuroligin 1 (NLGN1) belongs to the type-B carboxylesterase/lipase family, is a synaptic cell-adhesion molecule that is enriched in postsynaptic densities where it may recruit receptors, channels, and signal-transduction molecules to synaptic sites of cell adhesion. Neuroligins consists of five members (NLGN1, NLGN2, NLGN3, NLGN4, and NLGN4Y), which interact with beta-neurexins, and this interaction is involved in the formation of functional synapses. The extracellular domain of functional Neuroligin 1 associates as a dimer when analyzed by sedimentation equilibrium. Neuroligin 1 has a unique N-linked glycosylation pattern in the neuroligin family, and glycosylation and its processing modify neuroligin activity. Neuroligin 1 is a potent trigger for the de novo formation of synaptic connections, and it has recently been suggested that it is required for the maturation of functionally competent excitatory synapses. The persistent expression of Neuroligin 1 is required for the maintenance of NMDAR-mediated synaptic transmission, which enables the normal development of synaptic plasticity and long-term memory in the amygdala of adult animals.
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