Wnt proteins are secreted glycoproteins that contain a conserved pattern of 23-24 cysteine residues. Wnts play critical roles in both carcinogenesis and embryonic development.
Specifications and Use for R&D Wnt-3a
Source of R&D Wnt-3a
• A DNA sequence encoding the signal peptide from human CD33, Met 1 - Ala 16, joined with the mature mouse Wnt-3A, Ser 19 - Lys 352 (Accession # P27467; Roelink, H. and R. Nusse, 1991, Genes Dev. 5(3):381 - 388) was expressed in Chinese hamster ovary cells.
• Based on N-terminal amino acid sequencing, the mature recombinant mouse Wnt-3A starts at Ser 19. The mature protein consists of 334 amino acids and has a calculated molecular mass of approximately 37 kDa. As a result of glycosylation, the recombinant mouse Wnt-3A migrates as an approximately 41 kDa protein in SDS-PAGE under reducing conditions.
Purity of R&D Wnt 3a
• > 75%, as determined by SDS-PAGE and visualized by silver stain.
• < 1.0 EU per 1 μg of the cytokine as determined by the LAL method.
Activity of R&D Wnt-3a
• Measured by its ability to induce alkaline phosphatase synthesis by MC3T3E1 osteoblastic cells.
• The ED50 for this effect is typically 1 - 5 ng/mL.
• In an alternative assay, this protein also activates the β-Catenin reporter, TOPFLASH in HEK293T cells.
• Optimal concentrations should be determined by each laboratory for each application. Protein concentrations should be titrated based on cell type and if appropriate, passage number of the cell line.
• Lyophilized from a 0.2 μm filtered solution in PBS, 0.1 mM EDTA and up to 0.5% CHAPS containing 50 μg of bovine serum albumin per 1 μg of cytokine.
Reconstitution of R&D Wnt-3a
• It is recommended that sterile PBS containing at least 0.1% human serum albumin or bovine serum albumin be added to the vial to prepare a stock solution of 40 μg/mL.
Storage of R&D Wnt 3a
• Lyophilised samples are stable for up to twelve months from date of receipt at -20° C to -70° C.
• Upon reconstitution, this cytokine can be stored under sterile conditions at 2° - 8° C for one month or at -20° C to -70° C in a manual defrost freezer for three months without detectable loss of activity. • Avoid repeated freeze-thaw cycles.