WB result of NF-κB p65 Rabbit mAb
Primary antibody: NF-κB p65 Rabbit mAb at 1/1000 dilution
Lane 1: HeLa whole cell lysate 20 µg
Lane 2: Jurkat whole cell lysate 20 µg
Lane 3: MCF7 whole cell lysate 20 µg
Secondary antibody: Goat Anti-Rabbit IgG, (H+L), HRP conjugated at 1/10000 dilution
Predicted MW: 60 kDa
Observed MW: 60, 65 kDa
NF-κB p65 Recombinant Rabbit mAb (S-475-56)
NF-κB p65 Recombinant Rabbit mAb (S-475-56)
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Product Details
Product Details
Product Specification
| Host | Rabbit |
| Synonyms | Transcription factor p65, Nuclear factor NF-kappa-B p65 subunit, Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3, RELA, NFKB3 |
| Immunogen | Synthetic Peptide |
| Location | Cytoplasm, Nucleus |
| Accession | Q04206 |
| Clone Number | S-475-56 |
| Antibody Type | Recombinant mAb |
| Isotype | IgG |
| Application | WB, IHC-P, ICC, IP, ICFCM |
| Reactivity | Hu, Ms, Rt |
| Purification | Protein A |
| Concentration | 0.5 mg/ml |
| Conjugation | Unconjugated |
| Physical Appearance | Liquid |
| Storage Buffer | PBS, 40% Glycerol, 0.05% BSA, 0.03% Proclin 300 |
| Stability & Storage | 12 months from date of receipt / reconstitution, -20 °C as supplied |
Dilution
| application | dilution | species |
| WB | 1:1000 | |
| IP | 1:50 | |
| IHC-P | 1:500 | |
| ICC | 1:500 | |
| ICFCM | 1:500 |
Background
Transcription factor p65 also known as nuclear factor NF-kappa-B p65 subunit is a protein that in humans is encoded by the RELA gene. RELA, also known as p65, is a REL-associated protein involved in NF-κB heterodimer formation, nuclear translocation and activation. NF-κB is an essential transcription factor complex involved in all types of cellular processes, including cellular metabolism, chemotaxis, etc. Phosphorylation and acetylation of RELA are crucial post-translational modifications required for NF-κB activation. RELA has also been shown to modulate immune responses, and activation of RELA is positively associated with multiple types of cancer.
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Western Blot
WB result of NF-κB p65 Rabbit mAb
Primary antibody: NF-κB p65 Rabbit mAb at 1/1000 dilution
Lane 1: Neuro-2a whole cell lysate 20 µg
Secondary antibody: Goat Anti-Rabbit IgG, (H+L), HRP conjugated at 1/10000 dilution
Predicted MW: 60 kDa
Observed MW: 65 kDa
WB result of NF-κB p65 Rabbit mAb
Primary antibody: NF-κB p65 Rabbit mAb at 1/1000 dilution
Lane 1: PC-12 whole cell lysate 20 µg
Secondary antibody: Goat Anti-Rabbit IgG, (H+L), HRP conjugated at 1/10000 dilution
Predicted MW: 60 kDa
Observed MW: 65 kDa
FC
Flow cytometric analysis of 4% PFA fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling NF-κB p65 antibody at 1/500 dilution (0.1 μg)/ (Red) compared with a Rabbit monoclonal IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti - Rabbit IgG Alexa Fluor® 488 was used as the secondary antibody.
IP
NF-κB p65 Rabbit mAb at 1/50 dilution (1 µg) immunoprecipitating NF-κB p65 in 0.4 mg HeLa whole cell lysate.
Western blot was performed on the immunoprecipitate using NF-κB p65 Rabbit mAb at 1/1000 dilution.
Secondary antibody (HRP) for IP was used at 1/400 dilution.
Lane 1: HeLa whole cell lysate 20 µg (Input)
Lane 2: NF-κB p65 Rabbit mAb IP in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG IP in HeLa whole cell lysate
Predicted MW: 60 kDa
Observed MW: 65 kDa
Immunohistochemistry
IHC shows positive staining in paraffin-embedded human tonsil. Anti-NF-κB p65 antibody was used at 1/500 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded human breast cancer. Anti-NF-κB p65 antibody was used at 1/500 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded human lung adenocarcinoma. Anti-NF-κB p65 antibody was used at 1/500 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded human ovarian carcinoma. Anti-NF-κB p65 antibody was used at 1/500 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded mouse spleen. Anti-NF-κB p65 antibody was used at 1/500 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
IHC shows positive staining in paraffin-embedded rat spleen. Anti-NF-κB p65 antibody was used at 1/500 dilution, followed by a HRP Polymer for Mouse & Rabbit IgG (ready to use). Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris/EDTA buffer pH9.0 was performed before commencing with IHC staining protocol.
Immunocytochemistry
ICC analysis of HeLa cells treated with hTNFα (20ng/ml) for 20 mins (top panel) and HeLa cells untreated with hTNFα (20ng/ml) for 20 mins (below panel). Anti-NF-κB p65 antibody was used at 1/500 dilution (Green) and incubated overnight at 4°C. Goat polyclonal Antibody to Rabbit IgG - H&L (Alexa Fluor® 488) was used as secondary antibody at 1/1000 dilution. The cells were fixed with 4% PFA and permeabilized with 0.1% PBS-Triton X-100. Nuclei were counterstained with DAPI (Blue). Counterstain with tubulin (Red).
