Overview

  • Product name
    NFkB p65 Transcription Factor Assay Kit
    See all NF-kB p65 kits
  • Detection method
    Colorimetric
  • Sample type
    Nuclear Extracts
  • Assay type
    Semi-quantitative
  • Assay time
    3h 30m
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Product overview

    NFkB p65 Transcription Factor Assay Kit ab133112 is a non-radioactive, sensitive ELISA-based method for detecting specific transcription factor DNA binding activity in nuclear extracts.


    In the NFkB p65 assay, a double stranded DNA sequence containing the NFkB response element is immobilized onto the bottom of the wells of a 96-well plate. NFkB contained in a nuclear extract, binds to the NFkB response element, and is detected using an anti NFkB p65 antibody. A secondary antibody conjugated to HRP is added to provide a colorimetric readout at 450 nm.


    NFkB p65 transcription factor assay protocol summary:
    - prepare nuclear extracts from cells
    - add samples to wells
    - incubate for 1 hr or o/n
    - wash with wash buffer
    - add NFkB antibody and incubate for 1 hr , then wash
    - add HRP-conjugated secondary antibody and incubate for 1 hr, then wash
    - add developing solution and incubate for 15-45 min
    - add stop solution
    - analyze with microplate reader

  • Platform
    Microplate reader

Properties

  • Storage instructions
    Please refer to protocols.
  • Components 96 tests
    96-Well Plate Cover 1 unit
    Polysorbate 20 1 vial
    Transcription Factor Antibody Binding Buffer (10X) 1 x 3ml
    Transcription Factor Binding Assay Buffer (4X) 1 x 3ml
    Transcription Factor Developing Solution 1 x 12ml
    Transcription Factor Goat Anti-Rabbit HRP Conjugate 1 x 100µl
    Transcription Factor NFkB (Human p65) Positive Control 1 vial
    Transcription Factor NFkB (p65) Primary Antibody 1 vial
    Transcription Factor NF-kB 96-Well Strip Plate 1 unit
    Transcription Factor NFkB Competitor dsDNA 1 vial
    Transcription Factor Reagent A 1 x 120µl
    Transcription Factor Stop Solution 1 x 12ml
    Wash Buffer Concentrate (400X) 1 x 5ml
  • Research areas
  • Function
    NF-kappa-B is a pleiotropic transcription factor which is present in almost all cell types and is involved in many biological processed 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 and the heterodimeric p65-p50 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. 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. NF-kappa-B heterodimeric p65-p50 and p65-c-Rel complexes are transcriptional activators. The NF-kappa-B p65-p65 complex appears to be involved in invasin-mediated activation of IL-8 expression. The inhibitory effect of I-kappa-B upon NF-kappa-B the cytoplasm is exerted primarily through the interaction with p65. p65 shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. Associates with chromatin at the NF-kappa-B promoter region via association with DDX1.
  • Sequence similarities
    Contains 1 RHD (Rel-like) domain.
  • Domain
    the 9aaTAD motif is a transactivation domain present in a large number of yeast and animal transcription factors.
  • Post-translational
    modifications
    Ubiquitinated, leading to its proteasomal degradation. Degradation is required for termination of NF-kappa-B response.
    Monomethylated at Lys-310 by SETD6. Monomethylation at Lys-310 is recognized by the ANK repeats of EHMT1 and promotes the formation of repressed chromatin at target genes, leading to down-regulation of NF-kappa-B transcription factor activity. Phosphorylation at Ser-311 disrupts the interaction with EHMT1 without preventing monomethylation at Lys-310 and relieves the repression of target genes.
    Phosphorylation at Ser-311 disrupts the interaction with EHMT1 and promotes transcription factor activity (By similarity). Phosphorylation on Ser-536 stimulates acetylation on Lys-310 and interaction with CBP; the phosphorylated and acetylated forms show enhanced transcriptional activity.
    Reversibly acetylated; the acetylation seems to be mediated by CBP, the deacetylation by HDAC3. Acetylation at Lys-122 enhances DNA binding and impairs association with NFKBIA. Acetylation at Lys-310 is required for full transcriptional activity in the absence of effects on DNA binding and NFKBIA association. Acetylation can also lower DNA-binding and results in nuclear export. Interaction with BRMS1 promotes deacetylation of 'Lys-310'.
  • Cellular localization
    Nucleus. Cytoplasm. Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B). Colocalized with RELA in the nucleus upon TNF-alpha induction.
  • Information by UniProt
  • 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
    • v rel avian reticuloendotheliosis viral oncogene homolog A (nuclear factor of kappa light polypeptide gene enhancer in B cells 3 (p65))
    • 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
    see all
  • Database links

Images

  • After the treatment with LPS (10 μg/ml for 6 hrs), cells were lysed with hypotonic HEPES lysis buffer (pH 7.4) and centrifuged at 1000 g for 10 min at 4°C, supernatants were collected and used for the determination of intracellular p65- NF-κB by ELISA. The absorbance was read at 450 nm using spectrophotometer.

  • Jurkat cells were treated with PMA and ionomycin (+). Nuclear lysates were extracted (ab113474) and 40 uL, corresponding to 4e6 cells, were tested in duplicates (+/- SD).

  • Titration of positive control with or without inhibitor, background signal subtracted (duplicates; +/- SD).

  • Assay of cell lysates isolated from stimulated (20 ng/ml TNF alpha for 30 minutes) and nonstimulated HeLa cells demonstrating NFkB (p65) activity.

Protocols

References

This product has been referenced in:
  • Chen X  et al. Celastrol attenuates incision-induced inflammation and pain associated with inhibition of the NF-?B signalling pathway via SARM. Life Sci 205:136-144 (2018). Read more (PubMed: 29750991) »
  • Saber S  et al. Perindopril, fosinopril and losartan inhibited the progression of diethylnitrosamine-induced hepatocellular carcinoma in mice via the inactivation of nuclear transcription factor kappa-B. Toxicol Lett 295:32-40 (2018). Functional Studies . Read more (PubMed: 29859236) »
See all 15 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

For tissue samples, in place of steps 1 and 2 in the kit protocol part B. Purification of Cellular Nuclear Extracts, we recommend the following procedure with fresh tissue:

1. Weigh a fresh tissue sample and cut into very small pieces using a clean razor blade. Collect the pieces in a pre-chilled, clean Dounce homogenizer.

2. While keeping the sample on ice, add 3 mL of ice-cold 1X Hypotonic Buffer supplemented with DTT and Nonidet P-40 (3 uL of 1M DTT and 3 uL of 10% Nonidet P-40) per gram of tissue.

3. Homogenize the sample with a Dounce homogenizer or a polytron device and incubate on ice for 15 minutes.

4. Transfer to prechilled microcentrifuge tubes and centrifuge at 300 x g for 10 minutes at 4C.

Continue with step 4. in the protocol for ab133112.

As indicated in step B.10 of the protocol, following nuclear extraction you will keep a small aliquot of nuclear extract to quantitate the protein concentration, which you will use to normalize your results based on the total amount of input protein. Thus, we recommend using the recommended 10 uL of sample per well.

Although we have not isolated nuclear extracts from tissues for our transcription factor assay kits, other researchers have reported success from isolating nuclear extracts from tissue following the protocol above.

Read More
The samples came from young male mice frozen subcutaneous tissue. We got the nuclear extraction by Thermo nuclear extraction kit. The nuclear extraction protein concentration were not high. We removed the lipid layer after centrifuge in 4 degree.
The background and positive control OD value were very positive. The sample OD values were a little bit low. The reason may be that the tissue was not fresh the nuclear extraction kit may contain some salt that may interfere with the NFkB kit the nuclear protein concentration was too low.
We proceed the experiment as the protocol.
Overall the kit works well.

Abcam user community

Verified customer

Submitted May 15 2015

Answer

We recommend using 10 uL of sample per well. The nuclear extraction protocol in the kit results in the production of 100 uL of sample with approximately 50 ug of nuclear protein from 10^7 cells. If you are using a significantly smaller number of cells I recommend appropriately scaling the final resuspension volume in step 8 of the nuclear extraction protocol (for example, with 5^7 cells resuspend in 50 uL Complete Nuclear Extraction Buffer instead of 100 uL). The final volume of sample nuclear extract used per well should always be 10 uL as instructed in the protocol.

Read More

Answer


1- what is the benefit and meaning of Non-specific Binding Wells and Specific Competitor dsDNA Wells?
Answer: The non-specific binding wells will provide background data. The Specific competitor dsDNA wells involves addition of soluble p65 specific DNA target. In this well p65 will bind the soluble target DNA rather than the well-bound target DNA. This is another control well which illustrates what signal to expect if sample containg p65 is added but does not bind the well-bound dsDNA target DNA. This result illustrates specificity.
2- why there is a quantity of nfkb p65 positive control that is enough for 15 reactions although I need only to do 2 positive control wells?
Answer: Users may not wish to run all wells at the same time. So for example if you wish to run 4 separate assays, each testing 4 samples, you will need 8 positive control reaction wells.
3- There is no standard curve.How could I determine conc. of p65?
Answer: This is a qualitative assay rather than a quantitative assay. The results of this assay can be used to compare p65 levels between 2 samples, for example.

Read More

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up