Immunofluorescence analysis of Cathepsin D was performed using 70% confluent log phase T47D cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Cathepsin D (AF4G5) Mouse Monoclonal Antibody (MA517236) at 2ug/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Recombinant human protein purified from E.coli (His-Cathepsin D)|
|Storage buffer||HEPES with 0.15M NaCl, 0.01% BSA, 50% glycerol|
|Contains||0.03% sodium azide|
|Tested Applications||Dilution *|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Western Blot (WB)||0.2-1 µg/ml|
* 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.
A suggested positive control for this product is SK-BR3 cells.
Cathepsin D (CatD) is a ubiquitously expressed lysosomal aspartyl protease involved in the normal degradation of proteins apoptosis and autophagy. Human CatD is synthesized on the endoplasmic reticulum as a pre-pro-enzyme. Preprocathepsin D (412aa) is cleaved and glycosylated to form an inactive procathepsin D (392aa) and then further cleaved to generate an active intermediate (348aa) single-chain molecule. The active intermediate is further processed into mature two chain form of cathepsin D, this processing step is carried out by cathepsin B or L. The two-chain form consists of an amino terminal light chain and a carboxyl-terminal heavy chain. Additionally, several more amino acids are removed from the carboxyl terminus of the heavy chain. Procathepsin D (pCD), secreted from cancer cells, acts as a mitogen on both cancer and stromal cells and stimulates their proinvasive and pro-metastatic properties.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.