NF-kB p65 Recombinant Rabbit Monoclonal Antibody [PSH0-27]
cat.: HA721307
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat, Monkey
Applications: WB, IHC-P, IF-Cell, FC
Clonality: Monoclonal
Clone number: PSH0-27
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: PBS (pH7.4), 0.1% 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 502-551 / 551.
Positive control: HeLa cell lysate, NIH/3T3 cell lysate, PC-12 cell lysate, A431 cell lysate, Raji cell lysate, HL-60 cell lysate, Daudi cell lysate, COS-1 cell lysate, Mouse lung tissue lysate, human breast cancer tissue, human breast tissue, HeLa, NIH/3T3 cells treated with 50μg/mL TNF-α for 20 minutes, PC-12.
Subcellular location: Nucleus, Cytoplasm.
Recommended Dilutions:
  WB
  IHC-P
  IF-Cell
  FC
1:1,000
1:1,000
1:100-1:200
1:1,000
Uniprot #: SwissProt: Q04206 Human | Q04207 Mouse
Entrez Gene: 309165 Rat
Alternative names: Avian reticuloendotheliosis viral (v rel) oncogene homolog A MGC131774 NF kappa B p65delta3 nfkappabp65 NFkB p65 NFKB3 Nuclear factor kappaB 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 NFKB3 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
Images
HA721307_1.jpg Fig1: Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/500 dilution.

Lane 1: HeLa cell lysate (control) (35 µg/Lane)
Lane 2: HeLa-RELA-KO (1) (35 µg/Lane)
Lane 3: HeLa-RELA-KO (2) (35 µg/Lane)

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

Exposure time: 3 minutes;
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 (HA721307) at 1/500 dilution was used in 5% NFDM/TBST 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.
HA721307_2.jpg Fig2: Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/500 dilution.

Lane 1: HeLa cell lysate (10 µg/Lane)
Lane 2: NIH/3T3 cell lysate (10 µg/Lane)
Lane 3: PC-12 cell lysate (10 µg/Lane)
Lane 4: A431 cell lysate (10 µg/Lane)
Lane 5: Raji cell lysate (10 µg/Lane)
Lane 6: HL-60 cell lysate (10 µg/Lane)
Lane 7: Daudi cell lysate (10 µg/Lane)
Lane 8: COS-1 cell lysate (10 µg/Lane)
Lane 9: Mouse lung tissue lysate (20 µg/Lane)

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

Exposure time: 2 minutes;
8% 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 (HA721307) at 1/500 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/300,000 dilution was used for 1 hour at room temperature.
HA721307_3.jpg Fig3: Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/1,000 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 (HA721307) at 1/1,000 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.
HA721307_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded human breast tissue with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/1,000 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 (HA721307) at 1/1,000 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.
HA721307_5.jpg Fig5: Immunocytochemistry analysis of HeLa cells labeling NF-kB p65 with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, 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.
HA721307_6.jpg Fig6: Immunocytochemistry analysis of NIH/3T3 cells treated with or without 50μg/mL TNF-α for 20 minutes labeling NF-kB p65 with Rabbit anti-NF-kB p65 antibody (HA721307) at 1/200 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 (HA721307) at 1/200 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. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
HA721307_7.jpg Fig7: Flow cytometric analysis of HeLa cells labeling NF-kB p65.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA721307, 1/1,000) (red) 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).
HA721307_8.jpg Fig8: Flow cytometric analysis of NIH/3T3 cells labeling NF-kB p65.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA721307, 1ug/ml) (red) 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).
HA721307_9.jpg Fig9: Flow cytometric analysis of PC-12 cells labeling NF-kB p65.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA721307, 1/1,000) (red) 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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