Western blot analysis was performed on whole cell extracts (30 µglysate) of HeLa (Lane 1), HeLa treated with IFN gamma (100ng/ml IFN gamma for 72h) (Lane 2), THP-1 (Lane 3), THP-1 treated with PMA (50 ng PMA for 48h) and LPS ( 200 ng/ml LPS for 24 h)(Lane 4), U-937 (Lane 5), Raji (Lane 6) and Ramos (Lane 7).The blots were probed with PSMB9 Rabbit polyclonal Antibody (Product# PA1-1960, 2 µg/ml) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/ml, 1:2500 dilution). A 22 kDa band corresponding to PSMB9 was observed across the cell line tested. Apart from the desired band a non-specific band was also observed in untreated cells. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005)..
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Mixture of synthetic peptides corresponding to residues C R(207) V I L G N/D E L P K F Y D E(220) of human and mouse proteasome 20S LMP2.|
|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|
|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|
PA1-1960 detects proteasome 20S LMP2 from human and mouse cells.
PA1-1960 can be successfully used in Western blot procedures. By Western blot this antibody detects an ~23 kDa protein representing proteasome 20S LMP2 from mouse embryo fibroblast cell extract. This antibody also recognizes a band at ~24 kDa, which may correspond to LMP2 precursor protein.
The PA1-1960 immunogen is a mixture of synthetic peptides corresponding to residues C R(207) V I L G N/D E L P K F Y D E(220) of human and mouse proteasome 20S LMP2. These peptides are available for use in neutralization and control procedures.
The proteasome is a multicatalytic proteinase complex with a highly ordered ring-shaped 20S core structure. The core structure is composed of 4 rings of 28 non-identical subunits; 2 rings are composed of 7 alpha subunits and 2 rings are composed of 7 beta subunits. Proteasomes are distributed throughout eukaryotic cells at a high concentration and cleave peptides in an ATP/ubiquitin-dependent process in a non-lysosomal pathway. An essential function of a modified proteasome, the immunoproteasome, is the processing of class I MHC peptides. This gene encodes a member of the proteasome B-type family, also known as the T1B family, that is a 20S core beta subunit. This gene is located in the class II region of the MHC (major histocompatibility complex). Expression of this gene is induced by gamma interferon and this gene product replaces catalytic subunit 1 (proteasome beta 6 subunit) in the immunoproteasome. Proteolytic processing is required to generate a mature subunit. Two alternative transcripts encoding different isoforms have been identified; both isoforms are processed to yield the same mature subunit.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Inhibition of apoptosis in acute promyelocytic leukemia cells leads to increases in levels of oxidized protein and LMP2 immunoproteasome.
PA1-1960 was used in western blot to study the mechanism for the observed accumulation of oxidized proteins during aging and in age-related diseases.
|Khan MA,Oubrahim H,Stadtman ER||Proceedings of the National Academy of Sciences of the United States of America (101:11560)||2004|