Rockland

NFkB p65 Monoclonal Antibody (27F9.G4)

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NFkB p65 Antibody in Immunocytochemistry (ICC/IF) — Rockland Monoclonal anti NFKB p65 (Rel A) antibody was used to detect p65 by immunofluorescence at a dilution of 1:5000. Hela cells were grown to sub-confluent on 18 mm2 glass coverslips #1.5. Cells were either unstimulated (A), or stimulated (B) with 50 ng/mL of TNF alpha for 30 min prior fixation. Cells were then fixed in methanol and blocked with 10% normal goat serum (NGS), in PBS, and TritonX 0.2% (Tx) and incubated for 1 hr at RT with primary ab, counterstained with DAPI and washed in PBS/NGS/Tx. Cells were incubated for 1 hr at RT with Atto 425 conjugated anti mouse secondary antibody for STED CW imaging. Data was collected on a STED-CW TCS-SP5 Confocal system equipped with a DFC 350FX camera allowing sequential acquisition in widefield, confocal and STED CW imaging on the same system.

NFkB p65 Antibody in Immunocytochemistry (ICC/IF) — Anti NFkB monoclonal antibody - Immunocytochemistry. Tissue: Human Fibroblasts. Top: Before activation. Bottom: After activation with poly IC. Left: 600-401-271 Anti-p65 NLS specific - lot 18372. Right: 200-301-065 Anti-p65 C-Term monoclonal antibody - lot 26076. The two antibodies that are shown target different regions of the p65 protein. The different staining patterns are thought to correspond with different functional regions of the protein.

NFkB p65 Antibody in Immunohistochemistry (IHC) — Rockland Antibody 200-301-065 has been tested in immunohistochemistry, analyzed by an anatomic pathologist and validated for use in IHC applications against formalin-fixed, paraffin-embedded human tissues. showed moderate to strong staining within many tissues, including epithelium of the breast, small intestine, kidney, pancreas, prostate, skin, placenta, and uterus, as well as within neurons and lymphoid tissues such as spleen, thymus, and tonsil. The antibody produced an excellent signal with almost no background staining at a concentration of 2.5 µg/ml. The image displayed shows specific staining in colon carcinoma as the precipitated red signal, with a hematoxylin purple nuclear counterstain. Image provided courtesy of LifeSpan Biosciences, Seattle, WA

NFkB p65 Antibody in Western Blot (WB) — Rockland Monoclonal anti NFKB p65 (Rel A) antibody was used to detect p65 in b.end5 mouse endothelial cells. Unstimulated control cells (left) show cytoplasmic staining, TNF-alpha stimulated cells (right) show nuclear staining. For staining, cells were washed with PBS to remove all traces of culture media and fixed with paraformaldehyde 4 % (45 min). Slides were washed with PBT (phosphate buffer 0.1M + Triton-X-100 0.1 %), 3 times 10 min each, then, blocked with PBT + 5 % normal goat serum, 1 hour. Sample was Incubated overnight in primary antibody (1:600 in blocking buffer). After 3X wash in PBT for 10 min, slides were incubated 1 hour with secondary antibody (1:1000) and mounted in 1:1 PB glycerol. Images kindly provided by Tebu-Bio from Francisco Javier Carrillo-Salinas (PhD Student); Dra. Carmen Guaza Instituto Cajal (CSIC), Madrid, Spain

NFkB p65 Antibody in Western Blot (WB) — Rockland monoclonal anti p65 200-301-065 was used to detect p65 by Western blot. Samples were prepared in RIPA lysis buffer, boiled with NuPage 4x LDS Sample Buffer and run on NuPage 4-12% Bis-Tris Gels. Blot was incubated with primary antibody at a dilution of 1:500 and detected with HRP conjugated anti mouse antibody at a dilution of 1:10000. Image provided courtesy of Dr. Al Baldwin, University of North Carolina, Chapel Hill.

NFkB p65 Antibody in Western Blot (WB) — Rockland anti NFKB p65 (Rel A) monoclonal antibody 200-301-065 was used to detect ~65 kDa band (red arrow) in HeLa whole cell lysate. Lysate was run on 4-20% gradient gel transferred under standard conditions and blocked in 1% BSA-TTBS 30 min RT. Blot was probed with monoclonal anti p65 1:1000 in 1% BSA-TBS-T o/n 4°C and detected with HRP conjugated Rb-anti Mouse antibody 1:40,000 in MB-070 30 min RT.

NFkB p65 Antibody — PAGE-MAP (microsphere affinity proteomics) of Mouse Anti-NFKB p65 (Rel A) Antibody. (Catalog Number: 200-301-065, Lot Number: 26076). Antibody array western blot binding of gel free size separated fractions of multiple lysates (solid lines) and shotgun mass spectroscopy identification (dashed lines) of the target band run in parallel correlate confirming the specificity of this antibody against NFKB p65. Data was provided by the Lund-Johansen lab of Oslo University Hospital. For more information on PAGE-MAP/IP-MS identification of antibody specificity and its large-scale implementation for antibody validation see Sikorski et. al., (2018) Nature Methods 15, 909-912.

Product Details

200-301-065

Applications	Tested Dilution	Publications
Western Blot (WB)	1:1,000-1:5,000	-
Immunohistochemistry (Paraffin) (IHC (P))	1:200-1:600	-
Immunocytochemistry (ICC/IF)	Assay-dependent	-
Immunoprecipitation (IP)	1:1,000-1:5,000	-

Product Specifications
Species Reactivity	Human
Host/Isotype	Mouse / IgG2a, kappa
Class	Monoclonal
Type	Antibody
Clone	27F9.G4
Immunogen	NFkB p65 (Rel A) peptide corresponding to a region near the C-terminus of the human protein conjugated to Keyhole Limpet Hemocyanin (KLH). View antigen
Conjugate	Unconjugated
Form	Liquid
Concentration	1 mg/mL
Purification	Protein A
Storage buffer	0.02M potassium phosphate, pH 7.2, with 0.15M NaCl
Contains	0.01% sodium azide
Storage conditions	-20° C, Avoid Freeze/Thaw Cycles

Product Specific Information

Store vial at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.

Showed a single band by IEP (immunoelectrophoresis) when tested with anti-mouse antibody. Reactivity was confirmed by ELISA against peptide conjugated carrier protein and by Western blot against HeLa whole cell lysate.

Target Information

Nuclear factor kB p65 (NF-kB p65) is encoded by the RELA gene and is present on chromosome 11 in humans. NF-kB P65 is also known as RelA (v-rel avian reticuloendotheliosis viral oncogene homolog A) and belongs to the Rel family of proteins. It is one of the two subunits of NF-kB (Nuclear factor kappa-light-chain-enhancer of activated B cells) that heterodimerizes with the other subunits p50 or p52. However, binding of TNF alpha to its cognate receptor phosphorylates IKK which in turn phosphorylates I kB allowing proteasomal degradation of I kB. It specifically plays a key role in transcription of immunoglobulin k (kappa) gene in mature B-lymphoid cells.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

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Bioinformatics

Protein Aliases: MGC131774; NF KB; nf-kappa-b p65; NF-kappa-B p65delta3; NF-kappa-B transcription factor p65; NFkappaB p65; nfkb rela; NFkB-p50; NFKB1; nuclear factor; nuclear factor kappa b; Nuclear factor NF-kappa-B p65 subunit; Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3; REL A; Transcription factor p65; v-rel avian reticuloendotheliosis viral oncogene homolog A; v-rel reticuloendotheliosis viral oncogene homolog A

Gene Aliases: NFKB3; p65; RELA

UniProt ID: (Human) Q04206

Entrez Gene ID: (Human) 5970

Molecular Function: Rel homology transcription factor

negative regulation of transcription from RNA polymerase II promoter liver development hair follicle development stimulatory C-type lectin receptor signaling pathway acetaldehyde metabolic process transcription from RNA polymerase II promoter inflammatory response cellular defense response aging positive regulation of cell proliferation organ morphogenesis response to organic substance response to UV-B positive regulation of Schwann cell differentiation cytokine-mediated signaling pathway membrane protein intracellular domain proteolysis positive regulation of chondrocyte differentiation positive regulation of type I interferon production response to muramyl dipeptide response to progesterone response to insulin response to cobalamin cellular response to hepatocyte growth factor stimulus response to muscle stretch Fc-epsilon receptor signaling pathway negative regulation of protein catabolic process response to drug negative regulation of apoptotic process positive regulation of I-kappaB kinase/NF-kappaB signaling response to amino acid response to morphine positive regulation of interleukin-12 biosynthetic process negative regulation of transcription, DNA-templated positive regulation of transcription, DNA-templated positive regulation of transcription from RNA polymerase II promoter negative regulation of insulin receptor signaling pathway regulation of inflammatory response T cell receptor signaling pathway positive regulation of NF-kappaB transcription factor activity response to cAMP defense response to virus cellular response to hydrogen peroxide nucleotide-binding oligomerization domain containing 2 signaling pathway response to interleukin-1 cellular response to lipopolysaccharide cellular response to nicotine cellular response to interleukin-1 cellular response to interleukin-6 cellular response to tumor necrosis factor cellular response to peptide hormone stimulus negative regulation of NIK/NF-kappaB signaling positive regulation of miRNA metabolic process negative regulation of extrinsic apoptotic signaling pathway

Process(es)

RNA polymerase II regulatory region sequence-specific DNA binding RNA polymerase II core promoter proximal region sequence-specific DNA binding RNA polymerase II distal enhancer sequence-specific DNA binding transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding transcriptional repressor activity, RNA polymerase II core promoter proximal region sequence-specific binding transcriptional activator activity, RNA polymerase II distal enhancer sequence-specific binding DNA binding chromatin binding transcription factor activity, sequence-specific DNA binding transcription factor activity, RNA polymerase II distal enhancer sequence-specific binding protein binding transcription factor binding protein kinase binding chromatin DNA binding ubiquitin protein ligase binding protein complex binding activating transcription factor binding phosphate ion binding identical protein binding protein homodimerization activity actinin binding histone deacetylase binding transcription regulatory region DNA binding protein heterodimerization activity protein N-terminus binding NF-kappaB binding repressing transcription factor binding ankyrin repeat binding