Western blot analysis of uPAR was performed by loading 20µg of HS49 and U-251, known to express high levels of uPAR, and 20µg of LOVO whole cell lysate which lacks significant levels of uPAR onto a 12.5% Tris-HCl polyacrylamide gel under non-reducing conditions. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. Membranes were then probed with a rabbit polyclonal antibody recognizing uPAR (Product #PA3-001) at a dilution of 1:1500 overnight at 4°C on a rocking platform. Membranes were then washed in TBS-0.1% Tween 20 and probed with a goat anti-rabbit-HRP secondary antibody (Product # 31460) at a dilution of 1:15000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using Super Signal West Dura (Product #34075).
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Recombinant protein containing the D2D3 domain of human uPAR|
|Storage buffer||PBS with 30% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:1500|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
PA3-001 detects uPAR in human samples and has been successfully used in Western blot and immunoprecipitation applications.
The PA3-001 immunogen is a recombinant protein containing the D2D3 domain of human uPAR.
uPA binding to uPAR stimulates the interaction of uPAR with integral membrane proteins, such as integrins, which signal intracellularly to promote cytoskeletal reorganization and cell migration. The urokinase receptor (uPAR) consists of three internally disulfide-bonded domains and is attached to the cell surface by a glycosyl phosphatidylinositol (GPI) anchor. Upon cleavage of the GPI, uPAR is released from the plasma membrane into its soluble form. Both uPAR and soluble uPAR (suPAR) can be cleaved in the region that links domains D1 to D2 to yield a D1 and D2D3 fragment, which has direct chemotactic activity.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.