Caspase 2 Colorimetric Cell-Based ELISA Kit (CBCAB00564)

Product Name:	Caspase 2 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00564
ELISA Type:	Cell-Based
Target:	Caspase 2
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The Caspase 2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Caspase 2 protein expression profile in cells. The kit can be used for measuring the relative amounts of Caspase 2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Caspase 2.

Qualitative determination of Caspase 2 concentration is achieved by an indirect ELISA format. In essence, Caspase 2 is captured by Caspase 2-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: 835, UniProt ID: P42575, OMIM: 600639, Unigene: Hs.368982
Gene Symbol:	CASP2
Sub Type:	None

UniProt Protein Function:	CASP2: Involved in the activation cascade of caspases responsible for apoptosis execution. Might function by either activating some proteins required for cell death or inactivating proteins necessary for cell survival. Heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a p18 subunit and a p12 subunit. Interacts with LRDD. Expressed at higher levels in the embryonic lung, liver and kidney than in the heart and brain. In adults, higher level expression is seen in the placenta, lung, kidney, and pancreas than in the heart, brain, liver and skeletal muscle. Belongs to the peptidase C14A family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 3.4.22.55; Apoptosis; Protease Chromosomal Location of Human Ortholog: 7q34-q35 Cellular Component: membrane; mitochondrion; cytoplasm; nucleus; cytosol Molecular Function:protein domain specific binding; protein binding; enzyme binding; cysteine-type endopeptidase activity Biological Process: luteolysis; nerve growth factor receptor signaling pathway; DNA damage response, signal transduction resulting in induction of apoptosis; positive regulation of apoptosis; apoptosis; positive regulation of neuron apoptosis; germ cell programmed cell death; protein processing; brain development; proteolysis; regulation of caspase activity; DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest; aging
NCBI Summary:	This gene encodes a member of the cysteine-aspartic acid protease (caspase) family. Caspases mediate cellular apoptosis through the proteolytic cleavage of specific protein substrates. The encoded protein may function in stress-induced cell death pathways, cell cycle maintenance, and the suppression of tumorigenesis. Increased expression of this gene may play a role in neurodegenerative disorders including Alzheimer's disease, Huntington's disease and temporal lobe epilepsy. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Jan 2011]
UniProt Code:	P42575
NCBI GenInfo Identifier:	83300977
NCBI Gene ID:	835
NCBI Accession:	P42575.2
UniProt Secondary Accession:	P42575,P42576, Q59F21, Q7KZL6, Q86UJ3, Q9BUP7, Q9BZK9 Q9BZL0, A8K5F9, D3DXD6, E9PDN0,
UniProt Related Accession:	P42575
Molecular Weight:	10,309 Da
NCBI Full Name:	Caspase-2
NCBI Synonym Full Names:	caspase 2, apoptosis-related cysteine peptidase
NCBI Official Symbol:	CASP2
NCBI Official Synonym Symbols:	ICH1; NEDD2; CASP-2; NEDD-2; PPP1R57
NCBI Protein Information:	caspase-2; protease ICH-1; protein phosphatase 1, regulatory subunit 57; neural precursor cell expressed developmentally down-regulated protein 2
UniProt Protein Name:	Caspase-2
UniProt Synonym Protein Names:	Neural precursor cell expressed developmentally down-regulated protein 2; NEDD-2; Protease ICH-1Cleaved into the following 3 chains:Caspase-2 subunit p18; Caspase-2 subunit p13; Caspase-2 subunit p12
Protein Family:	Caspase
UniProt Gene Name:	CASP2
UniProt Entry Name:	CASP2_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-Caspase 2 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.)