Western blot analysis was performed on whole cell extracts (20 ug lysate) of NIH/3T3 (Lane 1), MCF7 (Lane 2), Hep G2 (Lane 3), Jurkat (lane 4) and HeLa (lane 5). The blots were probed with Anti-NF-kB p65 Rabbit Polyclonal Antibody (Product# PA1186, 1-3 ug/ml) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 ug/ml, 1:2500 dilution). A 60 kDa band corresponding to NF-kB p65 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), 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|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 30% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||1-5 µg x 10^6 cells|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||1-2 µg/ml|
|Immunofluorescence (IF)||1-2 µg/ml|
|Immunoprecipitation (IP)||3 µg|
|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|
PA1-186 has been successfully used in WB using StartingBlock T20 (TBS) Blocking Buffer (Product # 37543). More non-specific bands are observed when using 5% BSA for blocking.
By WB, PA1-186 detects a predominant band at 65kD. Loading increasing amounts of cell lysate may yield more intense nonspecific bands at ~120kD and/or ~50kD.
PA1-186 has been succesfully used for IP of the NF-kB p65 subunit and co-IP of the NF-kB p50 subunit.
NF-kB (nuclear factor kB) regulates the expression of a large number of genes that play critical roles in apoptosis, viral replication, tumorigenesis, various autoimmune diseases, and inflammation. The active nuclear form of the NF-kB transcription factor complex is composed of two DNA binding subunits, NF-kB p65 and p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65.
IP-MS enrichment of RELA (LFQ intensity): RELA was enriched 444-fold from A549 lysate compared to background proteins, using the optimized IP-MS workflow with Pierce MS-Compatible Magnetic IP Kit protein A/G (Part No. 90409) and RELA antibody (Part No. PA1-186). The STRING database (www.string-db.org) was used to identify the protein interactor list. See more information on IP-MS verification of antibody selectivity. IP-MS validation info.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
MicroRNA-27a Inhibits Cell Migration and Invasion of Fibroblast-Like Synoviocytes by Targeting Follistatin-Like Protein 1 in Rheumatoid Arthritis.
PA1-186 was used in western blot to investigate the effects of miR-27a on cell migration and invasion in fibroblast-like synoviocytes from rheumatoid arthritis patients
|Shi DL,Shi GR,Xie J,Du XZ,Yang H||Molecules and cells (39:611)||2016|