Immunofluorescence analysis of PSMA1 was performed using 70% confluent log phase MDA-MB-231 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 PSMA1 Rabbit Polyclonal Antibody (PA1963) at 2ug/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A27034) 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 control without primary antibody. The images were captured at 60X magnification.
|Tested species reactivity||Dog, Hamster, Human, Mouse, Rat|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic Peptide: C P(249) A D E P A E K A D E P M E H(263)|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Western Blot (WB)||1-3 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.
|Western Blot (WB)||See 1 publications below|
PA1-963 detects proteasome 20S C2 subunit from canine, hamster, human, mouse and rat tissues and cells.
PA1-963 has been successfully used in Western blot procedures. By Western blot, this antibody detects a 29 kDa protein representing proteasome 20S C2 subunit from rat brain extract.
PA1-963 immunizing peptide corresponds to amino acid residues 249-263 from the C-terminus of human proteasome 20S C2 subunit. This sequence is 93% conserved in the rat and chicken protein and 87% conserved in the mouse protein. PA1-963 immunizing peptide (Cat. # PEP-100) is available for use in neutralization and control experiments.
Proteolytic degradation is critical to the maintenance of appropriate levels of short-lived and regulatory proteins as important and diverse as those involved in cellular metabolism, heat shock and stress response, antigen presentation, modulation of cell surface receptors and ion channels, cell cycle regulation, transcription, and signalling factors. The ubiquitin-proteasome pathway deconstructs most proteins in the eukaryotic cell cytosol and nucleus. Others are degraded via the vacuolar pathway which includes endosomes, lysosomes, and the endoplasmic reticulum.
The 26S proteasome is an ATP-dependent, multisubunit (~31), barrel-shaped molecular machine with an apparent molecular weight of ~2.5 MDa. It consists of a 20S proteolytic core complex which is crowned at one or both ends by 19S regulatory subunit complexes. The 19S regulatory subunits recognize ubiquitinated proteins and play an essential role in unfolding and translocating targets into the lumen of the 20S subunit. An enzymatic cascade is responsible for the attachment of multiple ubiquitin molecules to lysine residues of proteins targeted for degradation. Several genetic diseases are associated with defects in the ubiquitin-proteasome pathway. Some examples of affected proteins include those linked to cystic fibrosis, Angelman and quote;s syndrome, and Liddle syndrome.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Glucosamine induces cell death via proteasome inhibition in human ALVA41 prostate cancer cell.
PA1-963 was used in western blot to investigate the mechanism for glucosamine-induced cell apoptosis in cancer cells
|Liu BQ,Meng X,Li C,Gao YY,Li N,Niu XF,Guan Y,Wang HQ||Experimental and molecular medicine (43:487)||2011|