NF-kB p65 Rabbit Polyclonal Antibody
cat.: HA500402
Product Type: Rabbit polyclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IHC-P, IF-Cell, FC, IP
Clonality: Polyclonal
Form: Liquid
Storage condition: Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.
Storage buffer: PBS (pH7.4), 0.1% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1.98ug/ul
Purification: Immunogen affinity purified.
Molecular weight: Predicted band size: 65 kDa
Isotype: IgG
Immunogen: Synthetic peptide within human RELA aa 450-550.
Positive control: HeLa cell lysate, MCF7 cell lysate, C2C12 cell lysate, RAW264.7 cell lysate, PC-12 cell lysate, C6 cell lysate, SH-SY5Y, PC-12, human breast tissue.
Subcellular location: Nucleus, Cytoplasm.
Recommended Dilutions:
  WB
  IHC-P
  IF-Cell
  FC
  IP
1:50,000
1:200
1:100-1:200
1:1,000
1-2μg/sample
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 NF-κB p65 NF κB ......
Images
HA500402_1.jpg Fig1: Western blot analysis of NF-kB p65 on different lysates with Rabbit anti-NF-kB p65 antibody (HA500402) at 1/50,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: MCF7 cell lysate
Lane 3: C2C12 cell lysate
Lane 4: RAW264.7 cell lysate
Lane 5: PC-12 cell lysate
Lane 6: C6 cell lysate

Lysates/proteins at 20 µg/Lane.

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

Exposure time: 59 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 (HA500402) at 1/50,000 dilution was used in primary antibody dilution (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.
HA500402_2.jpg Fig2: Immunocytochemistry analysis of SH-SY5Y cells labeling NF-kB p65 with Rabbit anti-NF-kB p65 antibody (HA500402) at 1/100 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 (HA500402) at 1/100 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.
HA500402_3.jpg Fig3: Immunocytochemistry analysis of PC-12 cells labeling NF-kB p65 with Rabbit anti-NF-kB p65 antibody (HA500402) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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 (HA500402) 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 (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.
HA500402_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded human breast tissue with Rabbit anti-NF-kB p65 antibody (HA500402) 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 (HA500402) 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.
HA500402_5.jpg Fig5: Flow cytometric analysis of SH-SY5Y cells labeling NF-kB p65.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA500402, 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).
HA500402_6.jpg Fig6: Flow cytometric analysis of PC-12 cells labeling NF-kB p65.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA500402, 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).
HA500402_7.jpg Fig7: NF-kB p65 was immunoprecipitated from 0.2 mg HeLa cell lysate with HA500402 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA500402 at 1/5,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HeLa cell lysate (input)
Lane 2: HA500402 IP in HeLa cell lysate
Lane 3: Rabbit IgG instead of HA500402 in HeLa cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 7 seconds; ECL: K1801
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.