HNF4alpha (Phospho-Ser313) Colorimetric Cell-Based ELISA Kit

Product Name:	HNF4alpha (Phospho-Ser313) Colorimetric Cell-Based ELISA
Product Code:	CBCAB01544
ELISA Type:	Cell-Based
Target:	HNF4alpha (Phospho-Ser313)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The HNF4alpha (Phospho-Ser313) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect HNF4alpha protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated HNF4alpha in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on HNF4alpha phosphorylation.

Qualitative determination of HNF4alpha (Phospho-Ser313) concentration is achieved by an indirect ELISA format. In essence, HNF4alpha (Phospho-Ser313) is captured by HNF4alpha (Phospho-Ser313)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ antibody can catalyze a colorimetric reaction upon substrate addition. 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 absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 3172, UniProt ID: P41235, OMIM: 125850/600281/606391, Unigene: Hs.116462
Gene Symbol:	HNF4A
Sub Type:	Phospho

UniProt Protein Function:	HNF4 alpha: a nuclear transcription factor which binds DNA as a homodimer. Controls the expression of several genes, including hepatocyte nuclear factor 1 alpha, a transcription factor which regulates the expression of several hepatic gene, alpha 1-antitrypsin, apolipoprotein CIII, and transthyretin genes. May be essential for development of the liver, kidney and intestine. Mutations have been associated with monogenic autosomal dominant non-insulin-dependent diabetes mellitus type I. Four alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:Nuclear receptor; DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 20q13.12 Cellular Component: nucleoplasm; cytoplasm; nucleus Molecular Function:RNA polymerase II transcription factor activity, enhancer binding; ligand-dependent nuclear receptor activity; protein binding; protein homodimerization activity; DNA binding; zinc ion binding; steroid hormone receptor activity; transcription factor activity; fatty acid binding; receptor binding Biological Process: transcription initiation from RNA polymerase II promoter; regulation of lipid metabolic process; intracellular receptor-mediated signaling pathway; positive regulation of transcription, DNA-dependent; lipid homeostasis; glucose homeostasis; endocrine pancreas development; regulation of transcription from RNA polymerase II promoter; negative regulation of cell proliferation; xenobiotic metabolic process; regulation of gastrulation; response to glucose stimulus; ornithine metabolic process; gene expression; positive regulation of transcription from RNA polymerase II promoter; steroid hormone mediated signaling; lipid metabolic process; negative regulation of cell growth; blood coagulation; regulation of insulin secretion; sex differentiation; phospholipid homeostasis Disease: Fanconi Renotubular Syndrome 4 With Maturity-onset Diabetes Of The Young; Maturity-onset Diabetes Of The Young, Type 1; Diabetes Mellitus, Noninsulin-dependent
NCBI Summary:	The protein encoded by this gene is a nuclear transcription factor which binds DNA as a homodimer. The encoded protein controls the expression of several genes, including hepatocyte nuclear factor 1 alpha, a transcription factor which regulates the expression of several hepatic genes. This gene may play a role in development of the liver, kidney, and intestines. Mutations in this gene have been associated with monogenic autosomal dominant non-insulin-dependent diabetes mellitus type I. Alternative splicing of this gene results in multiple transcript variants encoding several different isoforms. [provided by RefSeq, Apr 2012]
UniProt Code:	P41235
NCBI GenInfo Identifier:	148886624
NCBI Gene ID:	3172
NCBI Accession:	P41235.3
UniProt Secondary Accession:	P41235,O00659, O00723, Q14540, Q5QPB8, Q6B4V5, Q6B4V6 Q6B4V7, Q92653, Q92654, A5JW41, B2RPP8,
UniProt Related Accession:	P41235
Molecular Weight:
NCBI Full Name:	Hepatocyte nuclear factor 4-alpha
NCBI Synonym Full Names:	hepatocyte nuclear factor 4, alpha
NCBI Official Symbol:	HNF4A
NCBI Official Synonym Symbols:	TCF; HNF4; MODY; FRTS4; MODY1; NR2A1; TCF14; HNF4a7; HNF4a8; HNF4a9; NR2A21; HNF4alpha
NCBI Protein Information:	hepatocyte nuclear factor 4-alpha; TCF-14; HNF4alpha10/11/12; transcription factor 14; transcription factor HNF-4; hepatic nuclear factor 4 alpha; nuclear receptor subfamily 2 group A member 1
UniProt Protein Name:	Hepatocyte nuclear factor 4-alpha
UniProt Synonym Protein Names:	Nuclear receptor subfamily 2 group A member 1; Transcription factor 14; TCF-14; Transcription factor HNF-4
Protein Family:	Hepatocyte nuclear factor
UniProt Gene Name:	HNF4A
UniProt Entry Name:	HNF4A_HUMAN

Component	Quantity
96-Well Cell Culture Clear-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	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

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

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
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)
Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. 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 five minutes each time with gentle shaking on the orbital shaker. 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 5 minutes each time.
9.	Add 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 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-HNF4alpha (Phospho-Ser313) Antibody, Anti-HNF4alpha Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover 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 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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HNF4alpha (Phospho-Ser313) Colorimetric Cell-Based ELISA Kit

Description