- MA5-15870 - Invitrogen Antibodies NFKB1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1), U-87 MG (Lane 2), A549 (Lane 3), Jurkat (Lane 4), Raji (Lane 5) and HeLa (Lane 6). The blot was probed with Anti-NFkB p50 Monoclonal Antibody (Product # MA5-15870, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 50 kDa band corresponding to NFkB p50 was observed across the cell lines tested with an additional band at 105 kDa which is known to be the precursor of NFkB p50 called p105.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NFkB1was performed by loading 20 µg of indicated whole cell lysates and 7 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BX10). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% Milk in TBST for 1 hour at room temperature. NFkB1 was detected at ~50 kDa (p50) and ~105 kDa (p105) using a NFkB1 mouse monoclonal antibody (Product # MA5-15870, upper) at a dilution of 1:500 in blocking buffer overnight at 4°C on a rocking platform. GAPDH was detected using GAPDH rabbit polyclonal antibody (Product # PA1-987, lower), followed by a Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036) at a dilution of 1:2000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NFkB1was performed by loading 20 µg of nuclear (N) and cytoplasmic (C) A549 lysate fractions from cells untreated (-) and treated (+) with 20 ng/mL TNF alpha (Product # RTNFAI) for 30 minutes and 7 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BX10). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% Milk in TBST for 1 hour at room temperature. NFkB1 was detected at ~50 kDa (p50) and ~105 kDa (p105) using a NFkB1 mouse monoclonal antibody (Product # MA5-15870, upper) at a dilution of 1:500 in blocking buffer overnight at 4°C on a rocking platform. GAPDH was detected using GAPDH rabbit polyclonal antibody (Product # PA1-987, lower), followed by a Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036) at a dilution of 1:2000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NFkB p50 (Fig. a) was performed by loading 20 µg of HeLa Control (lane 1), HeLa NFkB p50 knockout (lane 2) whole cell extracts. NFkB p50 was detected at 50 kDa using NFkB p50 mouse monoclonal Antibody (Product # MA5-15870, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody HRP conjugate (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Loss of signal in CRISPR mediated knockout (KO) confirms that antibody is specific to NFkB p50.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of NFkB1 (green) in A549 cells either left untreated or treated 20ng/ml TNF-alpha for 20 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 3% BSA for 30 minutes at room temperature. Cells were stained with a NFkB1mouse monoclonal antibody (Product # MA5-15870) at a concentration of 5ug/mL in blocking buffer for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32731) at a dilution of 1:500 for at least 30 minutes at a room temperature in the dark (green). Nuclei (blue) were stained with Hoechst 33342 (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of NFkB1(green) in A431 cells. The cells were fixed with 100% ice cold methanol for 15 minutes at -20c, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 3% BSA for 30 minutes at room temperature. Cells were stained with a NFkB1mouse monoclonal antibody (Product # MA5-15870) at a concentration of 5 µg/mL in blocking buffer for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32731) at a dilution of 1:500 for at least 30 minutes at a room temperature in the dark (green). Nuclei (blue) were stained with Hoechst 33342 (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of NFkB1 (green) in A549 cells either left untreated or treated 20 ng/mL TNF-alpha for 20 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 3% BSA for 30 minutes at room temperature. Cells were stained with a NFkB1mouse monoclonal antibody (Product # MA5-15870) at a concentration of 5 µg/mL in blocking buffer for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32731) at a dilution of 1:500 for at least 30 minutes at a room temperature in the dark (green). Nuclei (blue) were stained with Hoechst 33342 (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometric analysis of MCF-7 cells using NFKB1 monoclonal antibody (Product # MA5-15870) (green) and negative control (purple).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Multiplex microplate Matrix ChIP has been described in detail (http://www.ncbi.nlm.nih.gov/pubmed/25959381). Briefly HTC116 cells were starved followed by addition of serum and samples of cells were cross-linked with formaldehyde after the time points indicated on the x-axis (0, 5 and 30 min). Chromatin was sheared using a Bioruptor and ChIP assays were performed using protein A-coated 96-well polypropylene microplates with 1uL/100uL well volume of NF kappa B monoclonal antibody (Product # MA5-15870). Quantitative real-time PCRs were performed in quadruplicate using 1 to 2uL of DNA with primers to -15kb downstream of Egr1 and exon 1 of Erg1. PCR calibration curves were generated for each primer pair from a dilution series of total human genomic DNA. The PCR primer efficiency curve was fit to cycle threshold (Ct) versus log [genomic DNA concentration] by using an r2 best fit. DNA concentration values for each ChIP and input DNA sample were calculated from their respective average Ct values. Final results are expressed as fraction of input DNA. Schematic representations of Erg1 loci is shown where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by the primers are represented by black bars. Data courtesy of Dr. Karol Bomsztyk’s laboratory

MA5-15870

Antibody data