Immunofluorescent analysis of Proteasome 19S Subunit S3 (green) showing staining in the cytoplasm and nucleus of NIH-3T3 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Proteasome 19S Subunit S3 polyclonal antibody (Product # PA1-974) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4°C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a flourescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Not Applicable|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic Peptide: E(513) R E Q Q D L E F A K E M A E D D D D S F P(534)|
|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)||1-20 µg/ml|
|Western Blot (WB)||1.0 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.
|Miscellaneous PubMed (MISC)||See 1 publications below|
PA1-974 detects proteasome 19S subunit S3 from human and mouse cells.
PA1-974 has been successfully used in ICC/IF, FACS, and Western blot procedures. By Western blot, this antibody detects an ~60 kDa protein representing proteasome 19S subunit S3 from HeLa cell extract.
For Western blot procedures it is necessary to transfer the protein to membrane at pH 11.
PA1-974 immunizing peptide corresponds to amino acid residues 513-534 from human proteasome 19S subunit S3 protein.
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.
|Not Applicable||Not Cited||
The cyclophilin-like domain of Ran-binding protein-2 modulates selectively the activity of the ubiquitin-proteasome system and protein biogenesis.
PA1-974 was used in western blot to investigate the role of proteasome subunits S10B during the proteolysis of intracellular proteins regulated by the ubiquitin-proteasome system.
|Yi H,Friedman JL,Ferreira PA||The Journal of biological chemistry (282:34770)||2007|