NF-kB p65 Recombinant Rabbit Monoclonal Antibody [SZ10-04]
cat.: ET1603-12
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat, Zebrafish
Applications: WB, IF-Cell, IF-Tissue, IHC-P, IP, FC
Clonality: Monoclonal
Clone number: SZ10-04
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 65 kDa
Isotype: IgG
Immunogen: Synthetic peptide within human RELA aa 490-540.
Positive control: A549 cell lysate, MCF7 cell lysate, HeLa cell lysate, RAW264.7 cell lysate, HeLa, human lung squamous cell carcinoma tissue, human lung cancer tissue, human lung tissue, human spleen tissue, mouse spleen tissue, rat spleen tissue.
Subcellular location: Nucleus, Cytoplasm.
Recommended Dilutions:
  WB
  IF-Cell
  IF-Tissue
  IHC-P
  FC
  IP
1:1,000-1:5,000
1:100-1:400
1:50-1:200
1:50-1:200
1:2,000
Use at an assay dependent concentration.
Uniprot #: SwissProt: Q04206 Human | Q04207 Mouse
Alternative names: Avian reticuloendotheliosis viral (v rel) oncogene homolog A MGC131774 NF kappa B p65delta3 NFKB3 Nuclear Factor NF Kappa B p65 Subunit Nuclear factor NF-kappa-B p65 subunit Nuclear factor of kappa light polypeptide gene enhancer in B cells 3 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3 OTTHUMP00000233473 OTTHUMP00000233474 OTTHUMP00000233475 OTTHUMP00000233476 OTTHUMP00000233900 p65 p65 NF kappaB p65 NFkB relA TF65_HUMAN Transcription factor p65 v rel avian reticuloendotheliosis viral oncogene homolog A (nuclear factor of kappa light polypeptide gene enhancer in B cells 3 (p65)) V rel avian reticuloendotheliosis viral oncogene homolog A v rel reticuloendotheliosis viral oncogene homolog A (avian) V rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light polypeptide gene enhancer in B cells 3, p65 NF-κB p65 NF κB p65 NF-κB p65
Images
ET1603-12_1.jpg Fig1: All lanes: Western blot analysis of NF-κB p65 with anti-NF-κB p65 antibody [SZ10-04] (ET1603-12) at 1:500 dilution.
Lane 1: Wild-type Hela whole cell lysate (20 µg).
Lane 2: NF-κB p65 knockout Hela whole cell lysate (20 µg).

ET1603-12 was shown to specifically react with NF-κB p65 in wild-type Hela cells. No band was observed when NF-κB p65 knockout sample was tested. Wild-type and NF-κB p65 knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1603-12, 1/500) and Loading control antibody (Rabbit anti-β-actin, R1207-1, 1/1,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG-HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.
ET1603-12_2.jpg Fig2: Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/1,000 dilution.

Lane 1: A549 cell lysate
Lane 2: MCF7 cell lysate
Lane 3: HeLa cell lysate
Lane 4: RAW264.7 cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 65 kDa
Observed band size: 65 kDa

Exposure time: 49 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1603-12) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.
ET1603-12_3.jpg Fig3: Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/2,000 dilution.

Lane 1: HAP1-parental cell lysate
Lane 2: HAP1-NF-kB p65 KD cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 65 kDa
Observed band size: 65 kDa

Exposure time: 60 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1603-12) at 1/2,000 dilution was used in K1803 at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
ET1603-12_4.jpg Fig4: Immunocytochemistry analysis of HeLa cells treated with or without 50 ng/mL TNF-alpha for 20 minutes labeling NF-kB p65 with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/400 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/400 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
ET1603-12_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded human lung squamous cell carcinoma tissue with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/400 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1603-12) at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1603-12_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded human lung cancer tissue with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1603-12) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1603-12_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded human lung tissue with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1603-12) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1603-12_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded human spleen tissue with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1603-12) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1603-12_9.jpg Fig9: Immunohistochemical analysis of paraffin-embedded mouse spleen tissue with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1603-12) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1603-12_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded rat spleen tissue with Rabbit anti-NF-kB p65 antibody (ET1603-12) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1603-12) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1603-12_11.jpg Fig11: NF-κB p65 was immunoprecipitated from 0.5 mg Hela whole cell lysates with ET1603-12 at 2 μg/mL. Western blot was performed from the immunoprecipitate using ET1603-12 at 1/500 dilution for 45 minutes at room temperature. Goat anti-Rabbit IgG-HRP Secondary Antibody (HA1001) was used at 1:300,000 dilution for 30 minutes at room temperature.

Lane 1: Hela whole cell lysates at 10 μg;
Lane 2: NF-κB p65 (ET1603-12) IP in Hela whole cell lysates;
Lane 3: Rabbit IgG instead of NF-κB p65 (ET1603-12) in Hela whole cell lysates.

Predicted band size: 60 kDa
Observed band size: 65 kDa

Exposure time: 10 seconds;

8% SDS-PAGE gel.
ET1603-12_12.jpg Fig12: Flow cytometric analysis of HeLa cells labeling NF-kB p65.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1603-12, red) at 1/2,000 dilution, compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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