NFkB p65 Antibody

SKU: orb344389

Description

NFkB p65 antibody

Images & Validation

• Tested Applications: ELISA, IF, IHC, WB
• Dilution range: ELISA: 1:50,000-1:100,000, IHC: 1:200-1:600, IF: 1:5,000, WB: 1:1,000-1:5,000
• Reactivity: Human
• Application Notes:
  Anti-NF-kB p65 Antibody is a mouse monoclonal antibody directed against NFkB p65 (Rel A) and recognizes a 65 kD band by Western blot against HeLa whole cell lysate. Control peptide (100-4165p) is sold separately. This product tested in WB, ICC, IHC, and IF.

Key Properties

• Antibody Type: Primary Antibody
• Host: Mouse
• Clonality: Monoclonal
• Isotype: IgG2a
• Clone No.: 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).
• Purity: Anti-NF-kB p65 Antibody was purified from concentrated tissue culture supernate by Protein A chromatography and 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.

Storage & Handling

• Storage: Store NF-kB p65 Antibody 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.
• Form/Appearance: Liquid (sterile filtered)
• Buffer/Preservatives: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
• Concentration: 0.98 mg/mL
• Dry Ice Shipping: Please note: This product requires shipment on dry ice. A dry ice surcharge will apply.
• Disclaimer: For research use only

Alternative Names

mouse anti-NF-kB p65 Antibody, mouse anti-Rel A antibody, NFKB, nfkb, NF-kB, NF-kappaB, NFkappaB, Nuclear factor NF-kappa-B p65 subunit

Anti NFkB monoclonal antibody – Immunocytochemistry. Tissue: Human Fibroblasts. Top: Before activation. Bottom: After activation with poly IC. Left: Anti-p65 NLS specific - (p/n orb345381). Right: Anti-p65 C-Term monoclonal antibody - (p/n orb344389). 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.

PAGE-MAP (microsphere affinity proteomics) of Mouse Anti-NFKB p65 (Rel A) Antibody. (orb344389). Antibody array western blot binding of gelfree 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.

Biorbyt anti NFKB p65 (Rel A) monoclonal antibody (p/n orb344389) was used to detect ~65 kD band (red arrow) in HeLa whole cell lysate (p/n orb348668). Lysate was run on 4-20% gradient gel transferred under standard conditions and blocked in 1% BSA-TBST for 30 min at RT. Blot was probed with monoclonal anti-NFkB p65 at 1:1000 in 1% BSA-TBST o/n at 4°C and detected with HRP conjugated Rb-anti-Mouse antibody (p/n orb347544) at 1:40000 in (p/n orb348637) for 30 min at RT.

Biorbyt Antibody (p/n orb344389) 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.

Biorbyt 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.

Biorbyt 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.

Quick Database Links

UniProt

Q04206

RefSeq:223468676

References

Deng, Dan et al. Silencing MEG3 protects PC12 cells from hypoxic injury by targeting miR-21 Artif Cells Nanomed Biotechnol, 48, 610-619 (2020)

PubMed: 32050796

Applications

WB

Reactivity

Rat

Yang, Xifeng et al. Downregulation of miR‑181b inhibits human colon cancer cell proliferation by targeting CYLD and inhibiting the NF‑κB signaling pathway Int J Mol Med, 46, 1755-1764 (2020)

PubMed: 32901872

Applications

WB

Reactivity

Human

Li, Ying et al. Pterostilbene pre-treatment reduces LPS-induced acute lung injury through activating NR4A1 Pharm Biol, 60, 394-403 (2022)

PubMed: 35271397

Applications

WB

Reactivity

Rat