This Mouse PSAP/Prosaposin overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of PSAP/Prosaposin protein (Cat: 51192-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the mouse Psap (NP_035309.3) (Met1-Asn557) was expressed with a polyhistidine tag at the C-terminus.
The recombinant mouse Psap consists 552 amino acids and predicts a molecular mass of 61.2 kDa.
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.
Prosaposin (PSAP) gene mutations, affecting saposin B (Sap-B) domain, cause a rare metachromatic leukodystrophy (MLD) variant in which arylsulfatase A (ARSA) activity is normal. The human prosaposin gene (PSAP) was previously localized to 10q21-->q22 by isotopic in situ hybridization using a human prosaposin cDNA as a probe. Using fluorescence in situ hybridization with a mouse genomic prosaposin fragment as probe, confirms the localization of PSAP and precisely maps it to band 10q22.1.