Western blot analysis was performed on whole cell extracts (30 ug lysate) of Jurkat (Lane 1), Jurkat treated with TNF alpha (Lane 2) . The blots were probed with ABfinity™ Rabbit Monoclonal Anti- NFkB p50 [pS337] Antibody (Product# 710460, 1-2 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 55 kDa band corresponding to NFkB p50 [pS337]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|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Peptide corresponding to amino acids 333–341 of human p50 [pS337]|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||1-3 ug x 10^6 cells|
|Western Blot (WB)||1-2 ug/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.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale and purified with Protein A.
ABfinity™ oligoclonal antibodies comprise a selection of multiple different recombinant monoclonal antibodies, providing the best of both worlds—the sensitivity of a polyclonal antibody with the specificity of a monoclonal, all delivered with the consistency only found in a recombinant antibody. While functionally the same as a polyclonal antibody—recognizing multiple epitope sites on the target and producing higher detection sensitivity for low abundance targets when compared with monoclonal antibodies—an oligoclonal antibody has a known mixture of light and heavy chains. This exact population can be produced in every lot, circumventing the biological variability typically associated with polyclonal antibody production.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
The p50 protein is a member of the NF- kappaB/Rel transcription factor family that is a subunit of the NF- kappaB protein complex. p50 is processed from a 105 kDa precursor protein by the 26S proteasome, and can exist as either a heterodimer or a homodimer. Heterodimers formed with Rel proteins activate transcription, but because p50 lacks a C terminal transactivation domain, the homodimer acts as a repressor unless it associates with a co-activator such as BCL3. The NF- kappaB protein complex expressed in most cell types, and is primarily found in the cytoplasm in an inactive state in association with I-kappa-B. Phosphorylation of I-kappa-B releases the complex, allowing translocation to the nucleus and modification of gene expression. Phosphorylation of the p50 subunit at the Ser-337 residue has been shown to significantly increase DNA binding efficiency of the NF- kappaB protein complex.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.