NFKB1 antibody (AA 860-969)

Catalog No. ABIN561981

Product Details anti-NFKB1 Antibody

Target: NFKB1 (Nuclear Factor of kappa Light Polypeptide Gene Enhancer in B-Cells 1 (NFKB1))

Binding Specificity: AA 860-969

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This NFKB1 antibody is un-conjugated

Application: Western Blotting (WB), ELISA, Immunofluorescence (IF), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Proximity Ligation Assay (PLA)

Purpose: Mouse monoclonal antibody raised against a partial recombinant NFKB1.

Sequence: MDNYEVSGGT VRELVEALRQ MGYTEAIEVI QAASSPVKTT SQAHSLPLSP ASTRQQIDEL RDSDSVCDSG VETSFRKLSF TESLTSGASL LTLNKMPHDY GQEGPLEGKI

Cross-Reactivity: Human

Characteristics: Antibody Reactive Against Recombinant Protein.

Immunogen: NFKB1 (AAH51765, 860 a.a. ~ 969 a.a) partial recombinant protein with GST tag. MW of the GST tag alone is 26 KDa.

Clone: 4A11

Isotype: IgG1

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Restrictions: For Research Use only

Independent Validation (IF)

Validation #104071 (Immunofluorescence)

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

No.: #104071

Date: 11/21/2019

Antigen: NFKB1

Lot Number: 11067-4A11

Method validated: Immunofluorescence

Positive Control:

U-937 human monocytic cell line

Negative Control:

No primary antibody control

Notes:

Passed. ABIN561981 specifically stains NF-κB1 in U-937 human monocytic cells.

Validation Images

U937 monocytic cells stained with with anti-NF-κB1/p50 antibody ABIN561981 and an AF488-labeled secondary antibody (bottom row, green). Counterstain with DAPI (blue). Negative control without primary antibody (top row) shows no AF488 signal.

Full Methods

Primary Antibody: ABIN561981

Secondary Antibody: 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.
• Block non-specific binding with 1x Duolink Blocking solution (Sigma-Aldrich, DUO82007) for 30min at RT.
• Incubate cells with primary mouse anti-human NF-κB1/p50 antibody (antibodies-online, ABIN561981, lot 11067-4A11) diluted 1:50 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.

Experimental Notes:

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.

Handling

Buffer: In 1x PBS, pH 7.4

Handling Advice: Aliquot to avoid repeated freezing and thawing.

Storage: -20 °C

Storage Comment: Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing.

Target Details for NFKB1

Target: NFKB1 (Nuclear Factor of kappa Light Polypeptide Gene Enhancer in B-Cells 1 (NFKB1))

Alternative Name: NFKB1 (NFKB1 Products)

Background: Full Gene Name: nuclear factor of kappa light polypeptide gene enhancer in B-cells 1
Synonyms: DKFZp686C01211,EBP-1,KBF1,MGC54151,NF-kappa-B,NFKB-p105,NFKB-p50,p105,p50

Gene ID: 4790

Pathways: p53 Signaling, NF-kappaB Signaling, RTK Signaling, TCR Signaling, TLR Signaling, Fc-epsilon Receptor Signaling Pathway, Neurotrophin Signaling Pathway, Activation of Innate immune Response, Myometrial Relaxation and Contraction, Regulation of Carbohydrate Metabolic Process, Hepatitis C, Toll-Like Receptors Cascades, BCR Signaling, S100 Proteins