Calsenilin/KCNIP3 (Phospho-Ser63)Colorimetric Cell-Based ELISA Kit (CBCAB01442)

Product Name:	Calsenilin/KCNIP3 (Phospho-Ser63)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01442
ELISA Type:	Cell-Based
Target:	Calsenilin/KCNIP3 (Phospho-Ser63)
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Calsenilin/KCNIP3 (Phospho-Ser63) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Calsenilin/KCNIP3 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Calsenilin/KCNIP3 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Calsenilin/KCNIP3 phosphorylation.

Qualitative determination of Calsenilin/KCNIP3 (Phospho-Ser63) concentration is achieved by an indirect ELISA format. In essence, Calsenilin/KCNIP3 (Phospho-Ser63) is captured by Calsenilin/KCNIP3 (Phospho-Ser63)-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: 30818, UniProt ID: Q9Y2W7, OMIM: 604662, Unigene: Hs.437376
Gene Symbol:	KCNIP3
Sub Type:	Phospho

UniProt Protein Function:	calsenilin: a small calcium-binding protein. Member of the family of voltage-gated potassium (Kv) channel-interacting proteins (KCNIPs), which belong to the recoverin branch of the EF-hand superfamily. Modulates K4 voltage-gated potassium channels. May regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium. Also functions as a calcium-regulated transcriptional repressor, and to interact with presenilins. May play a role in the regulation of PSEN2 proteolytic processing.
UniProt Protein Details:	Protein type:Transcription factor; Calcium-binding Chromosomal Location of Human Ortholog: 2q21.1 Cellular Component: Golgi apparatus; voltage-gated potassium channel complex; endoplasmic reticulum; dendrite; nerve terminal; nucleus; cytosol Molecular Function:protein C-terminus binding; potassium channel regulator activity; potassium channel activity; DNA binding; sequence-specific DNA binding; voltage-gated ion channel activity; calcium ion binding; calcium-dependent protein binding; transcription corepressor activity Biological Process: regulation of transcription from RNA polymerase II promoter; intracellular protein transport; behavioral response to pain; transcription, DNA-dependent; apoptosis; regulation of neuron apoptosis; negative regulation of transcription from RNA polymerase II promoter; sensory perception of pain; signal transduction
NCBI Summary:	This gene encodes a member of the family of voltage-gated potassium (Kv) channel-interacting proteins, which belong to the recoverin branch of the EF-hand superfamily. Members of this family are small calcium binding proteins containing EF-hand-like domains. They are integral subunit components of native Kv4 channel complexes that may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium. The encoded protein also functions as a calcium-regulated transcriptional repressor, and interacts with presenilins. Alternatively spliced transcript variants encoding different isoforms have been described. [provided by RefSeq, Jul 2008]
UniProt Code:	Q9Y2W7
NCBI GenInfo Identifier:	13431428
NCBI Gene ID:	30818
NCBI Accession:	Q9Y2W7.1
UniProt Secondary Accession:	Q9Y2W7,Q3YAC3, Q3YAC4, Q53TJ5, Q96T40, Q9UJ84, Q9UJ85 H7BY46,
UniProt Related Accession:	Q9Y2W7
Molecular Weight:	26,335 Da
NCBI Full Name:	Calsenilin
NCBI Synonym Full Names:	Kv channel interacting protein 3, calsenilin
NCBI Official Symbol:	KCNIP3
NCBI Official Synonym Symbols:	CSEN; DREAM; KCHIP3
NCBI Protein Information:	calsenilin; DRE-antagonist modulator; kv channel-interacting protein 3; potassium channel interacting protein 3; A-type potassium channel modulatory protein 3; calsenilin, presenilin-binding protein, EF hand transcription factor
UniProt Protein Name:	Calsenilin
UniProt Synonym Protein Names:	A-type potassium channel modulatory protein 3; DRE-antagonist modulator; DREAM; Kv channel-interacting protein 3; KChIP3
Protein Family:	Calsenilin
UniProt Gene Name:	KCNIP3
UniProt Entry Name:	CSEN_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-Calsenilin/KCNIP3 (Phospho-Ser63) Antibody, Anti-Calsenilin/KCNIP3 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.)