| Mouse Wnt-2b (also Wnt-13) is a 389 amino acid, 42kD member of the large and highly conserved Wnt family of secreted, cysteine-rich glycoprotein signaling molecules. Wnt proteins are associated with developmental and carcinogenic processes. Wnt-2b may be necessary to maintain stem cells in an undifferentiated state. As a type II transmembrane protein with a long C-terminal extracellular and a short N-terminal cytoplasmic domain, Wnt-2b is presumably cleaved within its extracellular region to release the secreted form. In the region from aa 64-104, mouse Wnt-2b is 100% identical to the human and rat protein, and over the 251-312 aa region also used as immunogen, mouse Wnt-2b is 97% and 100% identical to the corresponding human and rat sequences, respectively. Applications: Western Blot: This antibody can be used at 0.1-0.2ug/ml with the appropriate secondary reagents to detect Wnt-2b. The detection limit for rmWnt-2b is approximately 5 ng/lane and 1 ng/lane under non-reducing and reducing conditions, respectively. In this format, this antibody shows approximately 20% cross-reactivity with rhWnt-2, and less than 5% cross-reactivity with rhWnt-7a, -9a, rmWnt-1, -3a, -4, -5a, -5b, -8a, -8b, -9b, -10a, -10b, -11, -16. Immunocytochemistry: This antibody has been used at a concentration of 10ug/ml to detect Wnt-2b in BT20 human breast carcinoma cells. Cells were fixed with PBS containing 4% paraformaldehyde for 20 minutes at room temperature and blocked with PBS containing 10% normal donkey serum, 0.1% Triton X-100, and 1% BSA for 45 minutes at room temperature. After blocking, cells were incubated with diluted primary antibody overnight at 4 C followed by NorthernLightsTM-557-coupled anti-goat IgG at room temperature in the dark for one hour. Between each step, cells were washed with PBS containing BSA. Direct ELISA: This antibody can be used at 0.5-1.0ug/ml with the appropriate secondary reagents to detect mouse Wnt-2b. The detection limit for rmWnt-2b is approximately 0.5 ng/well. Optimal dilutions should be determined by each laboratory for each application. Reconstitution: Reconstitute with sterile PBS. If 0.5 mL of PBS is used, the antibody concentration will be 0.2 mg/mL. Storage and Stability: Lyophilized powder may be stored at 4C for short-term only. Reconstitute to nominal volume by adding sterile 40-50% glycerol and store at -20C. Reconstituted product is stable for 12 months at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. |
