ANKRD1 Colorimetric Cell-Based ELISA (CBCAB01106)

Product Name:	ANKRD1 Colorimetric Cell-Based ELISA
Product Code:	CBCAB01106
ELISA Type:	Cell-Based
Target:	ANKRD1
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The ANKRD1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ANKRD1 protein expression profile in cells. The kit can be used for measuring the relative amounts of ANKRD1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on ANKRD1.

Qualitative determination of ANKRD1 concentration is achieved by an indirect ELISA format. In essence, ANKRD1 is captured by ANKRD1-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 27063, UniProt ID: Q15327, OMIM: 106700/609599, Unigene: Hs.448589
Gene Symbol:	ANKRD1
Sub Type:	None

UniProt Protein Function:	ANKRD1: May play an important role in endothelial cell activation. May act as a nuclear transcription factor that negatively regulates the expression of cardiac genes. Induction seems to be correlated with apoptotic cell death in hepatoma cells. Defects in ANKRD1 may be a cause of total anomalous pulmonary venous return (TAPVR). TAPVR is a rare congenital heart disease (CHD) in which the pulmonary veins fail to connect to the left atrium during cardiac development, draining instead into either the right atrium or one of its venous tributaries. This disease accounts for 1.5% of all CHDs and has a prevalence of approximately 1 out of 15'000 live births.
UniProt Protein Details:	Protein type:Transcription factor Chromosomal Location of Human Ortholog: 10q23.31 Cellular Component: cytoplasm; cytosol; I band; nucleolus; nucleoplasm; nucleus; sarcomere Molecular Function:DNA binding; histone deacetylase binding; p53 binding; protein binding; titin binding; transcription corepressor activity Biological Process: cardiac muscle morphogensis; muscle cell differentiation; positive regulation of apoptosis; positive regulation of DNA damage response, signal transduction by p53 class mediator; positive regulation of protein secretion
NCBI Summary:	The protein encoded by this gene is localized to the nucleus of endothelial cells and is induced by IL-1 and TNF-alpha stimulation. Studies in rat cardiomyocytes suggest that this gene functions as a transcription factor. Interactions between this protein and the sarcomeric proteins myopalladin and titin suggest that it may also be involved in the myofibrillar stretch-sensor system. [provided by RefSeq, Jul 2008]
UniProt Code:	Q15327
NCBI GenInfo Identifier:	109940213
NCBI Gene ID:	27063
NCBI Accession:	Q15327.2
UniProt Secondary Accession:	Q15327,Q96LE7,
UniProt Related Accession:	Q15327
Molecular Weight:	36,252 Da
NCBI Full Name:	Ankyrin repeat domain-containing protein 1
NCBI Synonym Full Names:	ankyrin repeat domain 1
NCBI Official Symbol:	ANKRD1
NCBI Official Synonym Symbols:	ALRP; CARP; C-193; CVARP; MCARP; bA320F15.2
NCBI Protein Information:	ankyrin repeat domain-containing protein 1
UniProt Protein Name:	Ankyrin repeat domain-containing protein 1
UniProt Synonym Protein Names:	Cardiac ankyrin repeat protein; Cytokine-inducible gene C-193 protein; Cytokine-inducible nuclear protein
Protein Family:	Ankyrin repeat domain-containing protein
UniProt Gene Name:	ANKRD1
UniProt Entry Name:	ANKR1_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-ANKRD1 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.)