Name: Anti-NF-kB p65 antibody [E379]
SKU: ab32536
Rating: 4 (15 reviews)

Anti-NF-kB p65 antibody [E379] is a rabbit recombinant monoclonal antibody that is used to detect NF-kB p65 in ICC/IF, IHC-P, IP, Western blot. Suitable for Human, Mouse samples.

- Using biophysical QC, antibody identity is confirmed at a molecular level for unrivaled batch-batch consistency
- Specificity confirmed with RELA knockout cell line validation
- Validated on the Leica BOND™ RX automated IHC staining platform
- Cited in over 430 publications

Related conjugates and formulations

ab190589
Conjugated
Alexa Fluor® 647 Anti-NF-kB p65 antibody [E379]
ab205823
Conjugated
Biotin Anti-NF-kB p65 antibody [E379]
ab208750
Conjugated
PE Anti-NF-kB p65 antibody [E379]
ab207297
Carrier free
Anti-NF-kB p65 antibody [E379] - BSA and Azide free
ab190205
Conjugated
Alexa Fluor® 488 Anti-NF-kB p65 antibody [E379]
ab214846
Conjugated
Alexa Fluor® 594 Anti-NF-kB p65 antibody [E379]

Images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (AB32536), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.21% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Abcam Recommends

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	IHC-P	IP	Flow Cyt	WB	ICC/IF
Human	Tested	Tested	Not recommended	Tested	Tested
Mouse	Tested	Not recommended	Not recommended	Tested	Not recommended
Rat	Not recommended	Not recommended	Not recommended	Not recommended	Not recommended

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/5000	Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Species Human	Dilution info 1/5000	Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Rat	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/30	Notes For unpurified use at 1/200. We do not guarantee IP for mouse.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Mouse	Dilution info -	Notes For unpurified use at 1/200. We do not guarantee IP for mouse.
Species Rat	Dilution info -	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Mouse	Dilution info -	Notes Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. For unpurified use at 1/100.
Species Human	Dilution info -	Notes Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. For unpurified use at 1/100.
Species Rat	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/1000 - 1/100000	Notes This antibody might not detect target band in mouse tissues.
Species Human	Dilution info 1/1000 - 1/100000	Notes This antibody might not detect target band in mouse tissues.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Rat	Dilution info -	Notes This antibody might not detect target band in mouse tissues.

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/100	Notes For unpurified use at 1/250 -1/500.

Not recommended
Not recommended

Species	Dilution info	Notes
Species Mouse	Dilution info -	Notes For unpurified use at 1/250 -1/500.
Species Rat	Dilution info -	Notes -

Associated Products

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Target data

See full target information RELA

Function

NF-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52. The heterodimeric RELA-NFKB1 complex appears to be most abundant one. The dimers bind at kappa-B sites in the DNA of their target genes and the individual dimers have distinct preferences for different kappa-B sites that they can bind with distinguishable affinity and specificity. Different dimer combinations act as transcriptional activators or repressors, respectively. The NF-kappa-B heterodimeric RELA-NFKB1 and RELA-REL complexes, for instance, function as transcriptional activators. NF-kappa-B is controlled by various mechanisms of post-translational modification and subcellular compartmentalization as well as by interactions with other cofactors or corepressors. NF-kappa-B complexes are held in the cytoplasm in an inactive state complexed with members of the NF-kappa-B inhibitor (I-kappa-B) family. In a conventional activation pathway, I-kappa-B is phosphorylated by I-kappa-B kinases (IKKs) in response to different activators, subsequently degraded thus liberating the active NF-kappa-B complex which translocates to the nucleus. The inhibitory effect of I-kappa-B on NF-kappa-B through retention in the cytoplasm is exerted primarily through the interaction with RELA. RELA shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. Beside its activity as a direct transcriptional activator, it is also able to modulate promoters accessibility to transcription factors and thereby indirectly regulate gene expression. Associates with chromatin at the NF-kappa-B promoter region via association with DDX1. Essential for cytokine gene expression in T-cells (PubMed:15790681). The NF-kappa-B homodimeric RELA-RELA complex appears to be involved in invasin-mediated activation of IL-8 expression. Key transcription factor regulating the IFN response during SARS-CoV-2 infection (PubMed:33440148).

Alternative names

NFKB3, RELA, Transcription factor p65, Nuclear factor NF-kappa-B p65 subunit, Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.21% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: E379
Purification technique: Affinity purification Protein A
Specificity: This antibody recognises NF-kB p65. For WB, this antibody is unsuitable for detecting NF-KB p65 in mouse tissue lysates. The expression of NF-KB p65 is increased by lipopolysaccharides treatment reported by PMID: 18036230. Although some papers support the expression of NF-kB p65 in mouse tissue (PMID: 21479220 and 20008488), This antibody cannot detect band of interest in these mouse tissue.
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Abcam recommended secondaries - Goat Anti-Rabbit HRP (Goat Anti-Rabbit IgG H&L (HRP) ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077). Or search our wide range of secondary antibodies for use with your experiment.

Product Specifications
Anti-NF-kB p65 antibody [E379] (ab32536) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in ICC/IF, IHC-P, IP, WB in human, mouse samples.
Anti-NF-kB p65 antibody [E379] (ab32536) specifically detects NF-kB p65 (UniProt ID: Q04206; Molecular weight: 60kDa) and is sold in a convenient trial size to enable initial testing (20 µL) and larger sizes for subsequent scaling up experiments (100 µL and 1 mL).

Quality and Validation
Abcam's high quality manufacturing and validation processes ensure Anti-NF-kB p65 antibody [E379] (ab32536) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.
The specificity of Anti-NF-kB p65 antibody [E379] (ab32536) has been confirmed by testing in knockout samples.
Anti-NF-kB p65 antibody [E379] (ab32536) has been cited over 439 times in peer reviewed journals and is trusted by the scientific community.
Anti-NF-kB p65 antibody [E379] (ab32536) has 15 independent reviews from customers.

Related Products
Antibody clone E379 is also available pre-conjugated to a variety of labels for your convenience - Alexa Fluor® 488, Alexa Fluor® 647, Biotin, PE, Alexa Fluor® 594 (ab190205, ab190589, ab205823, ab208750, ab214846).

Patented technology
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

What are the advantages of a recombinant monoclonal antibody?
This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Species reactivity
Rat: We have preliminary internal testing data to indicate this antibody may not react with this species.
Please contact us for more information.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

19 product images

Souslova et al PLoS One. 2010 Mar 25;5(3):e9902. doi: 10.1371/journal.pone.0009902. Fig 2. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (ab32536)
MMP3 and NFκB are co-localized in human atherosclerotic plaques.
Sections (4 μm) of paraffin embedded tissue blocks of atherosclerotic plaques were subjected to double immunostaining of MMP3 and p50 (A, B, and C) or MMP3 and p65 (D, E and F, shown), using rabbit polyclonal NFκB p50 and MMP3 antibodies (Abcam) and ab32536, respectively, and goat anti-rabbit secondary antibodies.
The double immunostaining was visualized with Liquid Permanent Red chromogen and Vector Blue chromogen. Pink colour indicates expression of p50 (A, B and C) or p65 (D, E and F). Blue colour indicates expression of MMP3 (A, B, C, D, E and F.
Arrow indicates cell co-expressing MMP3 with p50 (B and C) or with p65 (E and F).
200× magnification in A and D; 400× magnification in B, C, E and F.
Fazalul Rahiman et al PLoS One. 2016 Apr 7;11(4):e0153005. doi: 10.1371/journal.pone.0153005. eCollection 2016. Fig 1. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] (ab32536)
NF-κB/p65 nuclear localisation in differentiated THP-1 (Human monocytic leukemia cell line) cells.
Cells were fixed, permeabilized, stained with ab32536 and visualised with Alexa Fluor^® 555 goat anti-rabbit IgG (green). The nuclei were counterstained with DAPI (blue). Microscopy images A. Inactive NF-κB/p65 proteins localization in the cytoplasm of the non-stimulated THP-1 cells (top row) and B. Translocated NF-κB/p65 proteins into the nuclei of THP-1 cells following LPS stimulation (bottom row).
Images were acquired for each fluorescence channel, using suitable TRITC and DAPI filters and a 63× objective.
Scale bar: 10μm
Immunoprecipitation - Anti-NF-kB p65 antibody [E379] (ab32536)
ab32536 (purified) at 1:30 dilution (2 μg) immunoprecipitating NF-kB p65 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.

Lane 1 (input): HeLa whole cell lysate 10μg
Lane 2 (+): ab32536 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32536 in HeLa whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.
All lanes: Immunoprecipitation - Anti-NF-kB p65 antibody [E379] (ab32536)
Predicted band size: 60 kDa
Western blot - Anti-NF-kB p65 antibody [E379] (ab32536)
Lane 1: Wild-type HAP1 cell lysate (20 μg)
Lane 2: NF-kB p65 knockout HAP1 cell lysate (20 μg)
Lane 3: HeLa cell lysate (20 μg)
Lane 4: A431 cell lysate (20 μg)
Lanes 1 - 4: Merged signal (red and green). Green - ab32536 observed at 70 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control ab8245 loading control, observed at 37 kDa.

Unpurified ab32536 was shown to specifically react with NF-kB p65 in wild-type HAP1 cells. No band was observed when NF-kB p65 knockout samples were used. Wild-type and NF-kB p65 knockout samples were subjected to SDS-PAGE. ab32536 (NF-kB p65) and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were diluted to 1/50 000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-NF-kB p65 antibody [E379] (ab32536)
Predicted band size: 60 kDa
Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] (ab32536)
Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling NF-kB p65 with purified ab32536 at 1:100 dilution.
Cells were fixed in 100% methanol. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain.
PBS instead of the primary antibody was used as the secondary antibody only control.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (ab32536)
Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling NF-kB p65 with ab32536 at 1/5000 (0.098 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on mouse spleen. The section was incubated with ab32536 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins
Western blot - Anti-NF-kB p65 antibody [E379] (ab32536)
Blocking/Diluting buffer and concentration: 5% NFDM/TBST
Exposure time: 37 second for Lane1 and 3 seconds for Lanes 2 and 3
Normal brain might express low level of p65 (PMID: 21479220)
All lanes: Western blot - Anti-NF-kB p65 antibody [E379] (ab32536) at 1/1000 dilution
Lane 1: Human fetal brain lysates at 20 µg
Lane 2: Human fetal kidney lysates at 20 µg
Lane 3: Human fetal lung lysates at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 60 kDa
Observed band size: 65 kDa
Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] (ab32536)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling NF-kB p65 with ab32536 at 1/100 (4.89 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing the signal translocated from the cytoplasm into the nucleus in NIH/3T3 cells after the treatment with TNF-alpha (50 ng/ml) for 20 min. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution in treated (right) and untreated cells (left).
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (ab32536)
Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling NF-kB p65 with ab32536 at 1/5000 (0.098 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human tonsil. The section was incubated with ab32536 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (ab32536)
Immunohistochemical analysis of paraffin-embedded Human prostatic hyperplasia tissue labeling NF-kB p65 with ab32536 at 1/5000 (0.098 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human prostatic hyperplasia. The section was incubated with ab32536 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (ab32536)
Immunohistochemical analysis of colon sections from mice, staining NF-kB p65 with unpurified ab32536.

Antigen retrieval was performed by microwave heating in citrate buffer, pH 6. Sections were incubated overnight with primary antibody (1/250) and staining was detected using ab80437 EXPOSE Rabbit specific HRP/DAB detection IHC kit.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (ab32536)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon carcinoma tissue sections labeling NF-kB p65 with Purified ab32536 at 1:2000 dilution (0.2 μg/ml).
Heat mediated antigen retrieval was performed using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used.
PBS instead of the primary antibody was used as the negative control.
Western blot - Anti-NF-kB p65 antibody [E379] (ab32536)
This antibody is unsuitable for detecting mouse tissue lysates.
The expression of NF-KB p65 is increased by lipopolysaccharides treatment reported by PMID: 18036230.
Although some papers support the expression of NF-κB p65 in mouse tissue (PMID: 21479220 and 20008488), ab32536 cannot detect band of interest in these mouse tissue.
All lanes: Western blot - Anti-NF-kB p65 antibody [E379] (ab32536) at 1000 µg
Lane 1: Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 15 µg
Lane 2: Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1ug/ml lipopolysaccharides for 6 hours whole cell lysates at 15 µg
Lane 3: Mouse brain lysates at 15 µg
Lane 4: Mouse spleen lysates at 15 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 60 kDa
Exposure time: 3s
Image courtesy of an anonymous customer review
Western blot - Anti-NF-kB p65 antibody [E379] (ab32536)
Anti-NF-kB p65 [E379] antibody (unpurified ab32536) reactivity with reduced wild type (WT) and p65 knockout (KO) mouse embryonic fibroblast (MEF) lysate.
After SDS-PAGE, membranes were blocked in 5% milk in TBS + 0.1% Tween for 1 hour at 25°C before incubation with unpurified ab32536 (1:1,000 dilution in 5% milk TBS + 0.1% Tween) for 16 hours at 4°C. Blots was then incubated with an anti-Rabbit HRP-conjugated secondary antibody before developing with ECL.
All lanes: Western blot - Anti-NF-kB p65 antibody [E379] (ab32536)
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 48 kDa, 60 kDa
Observed band size: 50 kDa, 58 kDa, 65 kDa, ~90 kDa
Exposure time: 30s
Image from Ali Ahmed Atef Ahmed et al. PLoS ONE 11.4 (2016): e0154278. Fig 8. doi: 10.1371/journal.pone.0154278.
Immunocytochemistry/ Immunofluorescence - Anti-NF-kB p65 antibody [E379] (ab32536)
Immunocytochemistry/ Immunofluorescence analysis of human cancer cells labeling NF-kB p65 with unpurified ab32536 (Middle panel).
Briefly, the tested cells were seeded on coverslips treated with HCl and ethanol, and autoclaved prior to use. Immunostaining of the p65 subunit of NF-κB was done by permeabilizing the cells with Triton X-10, then by treating the cells with anti-NF-κB p65 rabbit monoclonal primary antibody [E379] (ab32536), followed by Alexa Fluor^® 488 Donkey anti-rabbit IgG secondary antibody. Nuclei of cells were stained with DAPI (Left panel).
Merge (Right panel).
Images were acquired using fluorescence microscope.
Western blot - Anti-NF-kB p65 antibody [E379] (ab32536)
Blocking and diluting buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-NF-kB p65 antibody [E379] (ab32536) at 1/5000 dilution
All lanes: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 60 kDa
Western blot - Anti-NF-kB p65 antibody [E379] (ab32536)
Blocking and diluting buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-NF-kB p65 antibody [E379] (ab32536) at 1/5000 dilution
All lanes: MEF (Mouse embryonic fibroblast (immortalized)) whole cell lysates at 15 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 33 kDa, 60 kDa
Western blot - Anti-NF-kB p65 antibody [E379] (ab32536)
All lanes: Western blot - Anti-NF-kB p65 antibody [E379] (ab32536) at 1/100000 dilution
All lanes: HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Predicted band size: 60 kDa
Observed band size: 65 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 antibody [E379] (ab32536)
Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling NF-kB p65 with ab32536 at a concentration of 0.05 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.
Anti-CD68 antibody [KP1] ab955 Anti-NF-kB p65 antibody [E379] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.
Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)