This Mouse Podoplanin overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Podoplanin protein (Cat: 50256-M03H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain (Met 1-Leu 141) of mouse PDPN (NP_034459.2) precursor was fused with the C-terminal polyhistidine-tagged Fc region of human IgG1 at the C-terminus.
The recombinant mouse PDPN/Fc is a disulfide-linked homodimer after removal of the signal peptide. The reduced monomer consists of 367 amino acids and has a predicted molecular mass of 40.6 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rmPDPN/Fc monomer is approximately 60-65 kDa due to 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.
Podoplanin, also known as PDPN, is a type-I integral membrane glycoprotein with diverse distribution in human tissues. The physiological function of this protein may be related to its mucin-type character. The homologous protein in other species has been described as a differentiation antigen and influenza-virus receptor. The specific function of this protein has not been determined. Alternatively spliced transcript variants encoding different isoforms have been identified.PDPN is a mucin-type glycoprotein negatively charged by extensive O-glycosylation and a high content of sialic acid, which expresses the adhesive property. It is selectively expressed in lymphatic endothelium as well as lymphangiomas, Kaposi sarcomas, and in a subset of angiosarcomas with probable lymphatic differentiation. PDPN may contribute to form odontoblastic fiber or function as the anchorage to the tooth development and in proliferating epithelial cells of cervical loop and apical bud. The intensity of podoplanin expression is negatively correlated with the expression of CD34 and factor VIII. Podoplanin would be useful as a diagnostic marker for epithelioid hemangioendothelioma in liver tumors.