NF-kB p65 Antikörper

Produktnummer ABIN6135887

Produktdetails anti-NF-kB p65 Antikörper

Target: NF-kB p65 (NFkBP65) (Nuclear Factor-kB p65 (NFkBP65))

Reaktivität: Human

Wirt: Maus

Klonalität: Monoklonal

Konjugat: Dieser NF-kB p65 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), Immunofluorescence (IF)

Kreuzreaktivität: Human, Maus, Ratte

Produktmerkmale: Monoclonal Antibodies

Immunogen: Recombinant protein of human NF-kB p65

Isotyp: IgG

Anwendungsinformationen

Applikationshinweise: WB,1:500 - 1:2000,IHC,1:50 - 1:200,IF,1:50 - 1:100,IP,1:20 - 1:50

Kommentare:

HIGH QUALITY

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (IF)

Validierung #104075 (Immunofluorescence)

by: Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie des Universitätsklinikums Knappschaftskrankenhaus Bochum

No.: #104075

Datum: 21.11.2019

Antigen: NfkBP65

Chargennummer: 9410609001

Validierte Anwendung: Immunofluorescence

Positivkontrolle:

U-937 human monocytic cell line

Negativkontrolle:

No primary antibody control

Bewertung:

Passed. ABIN6135887 specifically stains RelA/p65 in U-937 human monocytic cells with antigen retrieval using 1% SDS.

Validierungsbilder

U937 monocytic cells stained with with anti- RelA/p65 antibody ABIN6135887 and an AF488-labeled secondary antibody (bottom row, green). Counterstain with DAPI (blue). Negative control without primary antibody (top row) shows no AF488 signal.

Protokoll

Primärantikörper: ABIN6135887

Sekundärantikörper: AF488-conjugated AffiniPure alpaca anti-mouse IgG antibody (Jackson Immuno Research, 615-545-214)

Full Protocol:

• Grow U-937 cells in RPMI 1640 Medium (Gibco, 21875034) supplemented with 10 % FCS (Gibco, A3840001) and Pen/Strep (Gibco, 15140122), at 37°C and 5% CO₂ in 300µL in 24-well plates (Sarstedt AG&Co. KG).
• Use cytospin to spin cells onto slide.
• Fix cells on coverslide in 4% paraformaldehyde (Carl Roth GmbH, 0335.1) for 30min at RT.
• Wash cells 3x for 5min with PBS (Sigma-Aldrich, 8537).
• Permeabilize cells in 100µL PBS containing 0.1 % Triton X-100 (Sigma-Aldrich, T8787) for 5min at RT.
• Wash cells 3x for 5min with PBS.
• Permeablilize cells in 100 µL PBS containing 1 % SDS (0183, Carl Roth GmbH) for 5 min at RT.
• Wash cells 3x for 5 min with PBS.
• Block non-specific binding with 1x Duolink Blocking solution (Sigma-Aldrich, DUO82007) for 30min at RT.
• Incubate cells with primary mouse anti-human RelA/p65 antibody (antibodies-online, ABIN6135887, lot 9410609001) diluted 1:100 in 1x Duolink Antibody diluent (Sigma-Aldrich, DUO82008) ON at 4°C.
• Wash cells 3x for 5min with PBS.
• Incubate cells with secondary AF488-conjugated AffiniPure alpaca anti-mouse IgG antibody (Jackson Immuno Research, 615-545-214) diluted 1:400 in 1x Duolink Antibody diluent for 1h at RT in the dark.
• Wash cells 2x for 5min with PBS.
• Mount coverslips on glass slides in 50µL ProLong Gold antifade mountant with DAPI (Invitrogen, P36931).
• Image acquisition with an Olympus widefield microscope.

Anmerkungen:

Strong green staining dependent on the presence of primary antibody indicates that the antibody works. Negative control lacking the primary antibody shows only background fluorescence. Staining was obtained using antigen retrieval (1% SDS). Without this the antibody did not yield detectable signal.

Handhabung

Format: Liquid

Buffer: PBS with 0.02 % sodium azide,50 % glycerol, pH 7.3.

Konservierungsmittel: Sodium azide

Vorsichtsmaßnahmen: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Lagerung: -20 °C

Informationen zur Lagerung: Store at -20°C. Avoid freeze / thaw cycles.

Referenzen für anti-NF-kB p65 Antikörper (ABIN6135887)

Fang, Fang, Ren, Hong, Liu, Zhao, Wang, Peng, Xiao: "Porcine Deltacoronavirus Accessory Protein NS6 Antagonizes Interferon Beta Production by Interfering with the Binding of RIG-I/MDA5 to Double-Stranded RNA." in: Journal of virology, Vol. 92, Issue 15, (2018) (PubMed).

Details zu NF-kB p65

Target: NF-kB p65 (NFkBP65) (Nuclear Factor-kB p65 (NFkBP65))

Andere Bezeichnung: RELA (NFkBP65 Produkte)

Hintergrund: NF-kappa-B is a ubiquitous transcription factor involved in several biological processes. It is held in the cytoplasm in an inactive state by specific inhibitors. Upon degradation of the inhibitor, NF-kappa-B moves to the nucleus and activates transcription of specific genes. NF-kappa-B is composed of NFKB1 or NFKB2 bound to either REL, RELA, or RELB. The most abundant form of NF-kappa-B is NFKB1 complexed with the product of this gene, RELA. Four transcript variants encoding different isoforms have been found for this gene.,NFKB3,p65,NF-kB p65,RELA,CMCU,Epigenetics & Nuclear Signaling,Transcription Factors,Cancer,Signal Transduction,MAPK-P38 Signaling Pathway,Cell Biology & Developmental Biology,Apoptosis,Inhibition of Apoptosis,Death Receptor Signaling Pathway,Endocrine & Metabolism,Endocrine and metabolic diseases,Obesity,Immunology & Inflammation,B Cell Receptor Signaling Pathway,T Cell Receptor Signaling Pathway,IL-6 Receptor Signaling Pathway,NF-kB Signaling Pathway,Toll-like Receptor Signaling Pathway,Neuroscience,Neurodegenerative Diseases,Amyloid Plaque and Neurofibrillary Tangle Formation in Alzheimer's Disease,Cardiovascular,RELA

Molekulargewicht: 58 kDa/59 kDa/60 kDa

Gen-ID: 5970

UniProt: Q04206

Pathways: NF-kappaB Signalweg, RTK Signalweg, T-Zell Rezeptor Signalweg, TLR Signalweg, Fc-epsilon Rezeptor Signalübertragung, Neurotrophin Signalübertragung, Activation of Innate immune Response, Cellular Response to Molecule of Bacterial Origin, Hepatitis C, Toll-Like Receptors Cascades, S100 Proteine