Description

NF-kB p65 (Phospho-Ser529) Cell-Based ELISA Kit

The NF-kB p65 (Phospho-Ser529) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor NF-kB p65 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated NF-kB p65 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on NF-kB p65 phosphorylation.

How does our NF-kB p65 (Phospho-Ser529) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of NF-kB p65 (Phospho-Ser529) concentration is achieved by an indirect ELISA format. In essence, NF-kB p65 (Phospho-Ser529) is captured by NF-kB p65 (Phospho-Ser529)-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target RFU values.
2.	An antibody against the nonphosphorylated counterpart of NF-kB p65 (Phospho-Ser529) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated NF-kB p65 can be used to normalize the RFU value for phosphorylated NF-kB p65.

NF-kB p65 (Phospho-Ser529) Fluorometric Cell-Based ELISA Kit -Information

Product Name:	NF-kB p65 (Phospho-Ser529) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00019
Description:	The NF-kB p65 (Phospho-Ser529) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor NF-kB p65 (Phospho-Ser529) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated NF-kB p65 (Phospho-Ser529) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on RELA phosphorylation.
Dynamic Range:	> 5000 Cells
Detection Method:	Fluorometric
Storage/Stability:	4°C/6 Months
Reactivity:	Human, Mouse, Rat
Assay Type:	Cell-Based ELISA
Database Links:	Gene ID: 5970, UniProt ID: Q04206, OMIM #: 164014, Unigene #: Hs.502875
Format:	Two 96-Well Plates
NCBI Gene Symbol:	RELA
Sub Type:	Phospho
Target Name:	Phospho-NF-?B p65 (Ser529)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components	Quantity
96-Well Black Cell CultureClear-Bottom Microplate	2 plates
10X TBS	24 ml
Quenching Buffer	24 ml
Blocking Buffer	50 ml
15X Wash Buffer	50 ml
Primary Antibody Diluent	12 ml
100x Anti-Phospho Target Antibody	60 µl
100x Anti-Target Antibody	60 µl
Anti-GAPDH Antibody	110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody	6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody	6 ml
Adhesive Plate Seals	2 seals

Additional equipment and materials required

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
Micropipettes capable of measuring volumes from 1 µl to 1 ml
Deionized or sterile water (ddH2O)
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.	Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. For suspension cells and loosely attached cells, coat the plates with 100 µl of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.	Incubate the cells for overnight at 37°C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µl of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µl of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µl 1x Wash Buffer for 3 minutes. The plate can be stored at 4°C for a week.
7.	Add 100 µl of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 3 minutes each time.
9.	Dispense 200 µl of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
11.	Add 50 µl of Primary Antibody Mixture P to corresponding wells for NF-kB p65 (Phospho-Ser529) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total NF-kB p65 detection. Cover the plate with parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
13.	Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.	Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.	Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.	Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

NF-kB p65 (Phospho-Ser529) - Protein Information

UniProt Protein Function:	NFkB-p65: a subunit of NF-kappa-B transcription complex, which plays a crucial role in inflammatory and immune responses. The inhibitory effect of I-kappa-B upon NF-kappa-B in 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. There are five NFkB proteins in mammals (RelA/NFkB-p65, RelB, c-Rel, NF-_B1/NFkB-p105, and NF-_B2/NFkB-p100). They form a variety of homodimers and heterodimers, each of which activates its own characteristic set of genes. Three splice-variant isoforms have been identified.
UniProt Protein Details:	Protein type:DNA-binding; Nuclear receptor co-regulator; Transcription factor Chromosomal Location of Human Ortholog: 11q13.1 Cellular Component: cytoplasm; cytosol; nuclear chromatin; nucleoplasm; nucleus; transcription factor complex Molecular Function:actinin binding; chromatin binding; chromatin DNA binding; DNA binding; histone deacetylase binding; identical protein binding; NF-kappaB binding; phosphate binding; protein binding; protein heterodimerization activity; protein homodimerization activity; protein kinase binding; protein N-terminus binding; RNA polymerase II transcription factor activity, enhancer binding; transcription activator binding; transcription factor activity; transcription factor binding; ubiquitin protein ligase binding Biological Process: activation of NF-kappaB transcription factor; cytokine and chemokine mediated signaling pathway; inflammatory response; membrane protein intracellular domain proteolysis; negative regulation of apoptosis; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; positive regulation of cell proliferation; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of interferon type I production; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of inflammatory response; response to organic substance; response to UV-B; stimulatory C-type lectin receptor signaling pathway; T cell receptor signaling pathway
NCBI Summary:	NF-kappa-B is a ubiquitous transcription factor involved in several biological processes. It is held in the cytoplasm in an inactive state by specific inhibitors. Upon degradation of the inhibitor, NF-kappa-B moves to the nucleus and activates transcription of specific genes. NF-kappa-B is composed of NFKB1 or NFKB2 bound to either REL, RELA, or RELB. The most abundant form of NF-kappa-B is NFKB1 complexed with the product of this gene, RELA. Four transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2011]
UniProt Code:	Q04206
NCBI GenInfo Identifier:	62906901
NCBI Gene ID:	5970
NCBI Accession:	Q04206.2
UniProt Secondary Accession:	Q04206,Q6GTV1, Q6SLK1,
UniProt Related Accession:	Q04206
Molecular Weight:	59,910 Da
NCBI Full Name:	Transcription factor p65
NCBI Synonym Full Names:	RELA proto-oncogene, NF-kB subunit
NCBI Official Symbol:	RELA
NCBI Official Synonym Symbols:	p65; NFKB3
NCBI Protein Information:	transcription factor p65
UniProt Protein Name:	Transcription factor p65
UniProt Synonym Protein Names:	Nuclear factor NF-kappa-B p65 subunit; Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3
Protein Family:	Proline-rich P65 protein
UniProt Gene Name:	RELA