ILK Colorimetric Cell-Based ELISA Kit (CBCAB00181)

Product Name:	ILK Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00181
ELISA Type:	Cell-Based
Target:	ILK
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of ILK concentration is achieved by an indirect ELISA format. In essence, ILK is captured by ILK-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: 3611, UniProt ID: Q13418, OMIM: 602366, Unigene: Hs.5158/Hs.706355
Gene Symbol:	ILK
Sub Type:	None

UniProt Protein Function:	ILK: a tyrosine kinase-like kinase of the MLK family. Couples integrins and growth factors to downstream pathways involved in cell survival, cell cycle control, cell-cell adhesion and cell motility. Functions as a scaffold bridging the extra-cellular matrix and growth factor receptors to the actin cytoskeleton through interactions with the ILK-PINCH complex. This complex, which includes integrin, PINCH (which links ILK to receptor tyrosine kinases via Nck2), PARVA and affixin, is considered to be one of the convergence points of integrin- and growth factor-signaling pathways. May be implicated in mediating cell architecture, adhesion to integrin substrates and anchorage-dependent growth in epithelial cells. Stimulated downstream of PI3 kinase. Increased expression is correlated with progression of several tumor types, including breast, prostate, and colon carcinomas. Overexpression drives anchorage-independent growth and faster cell cycle.
UniProt Protein Details:	Protein type:Protein kinase, Ser/Thr (non-receptor); EC 2.7.11.1; Kinase, protein; Protein kinase, TKL; TKL group; MLK family; ILK subfamily Chromosomal Location of Human Ortholog: 11p15.4 Cellular Component: cell junction; cytoplasm; cytosol; focal adhesion; membrane; nucleoplasm Molecular Function:protein binding; protein kinase binding; protein serine/threonine kinase activity Biological Process: cell-matrix adhesion; integrin-mediated signaling pathway; positive regulation of phosphorylation; positive regulation of transcription, DNA-dependent; protein amino acid phosphorylation
NCBI Summary:	This gene encodes a protein with a kinase-like domain and four ankyrin-like repeats. The encoded protein associates at the cell membrane with the cytoplasmic domain of beta integrins, where it regulates integrin-mediated signal transduction. Activity of this protein is important in the epithelial to mesenchymal transition, and over-expression of this gene is implicated in tumor growth and metastasis. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jun 2013]
UniProt Code:	Q13418
NCBI GenInfo Identifier:	9973397
NCBI Gene ID:	3611
NCBI Accession:	Q13418.2
UniProt Secondary Accession:	Q13418,P57043, Q68DZ3, B7Z1I0, B7Z418, D3DQU0,
UniProt Related Accession:	Q13418
Molecular Weight:	36,467 Da
NCBI Full Name:	Integrin-linked protein kinase
NCBI Synonym Full Names:	integrin linked kinase
NCBI Official Symbol:	ILK
NCBI Official Synonym Symbols:	P59; ILK-1; ILK-2; p59ILK; HEL-S-28
NCBI Protein Information:	integrin-linked protein kinase
UniProt Protein Name:	Integrin-linked protein kinase
UniProt Synonym Protein Names:	59 kDa serine/threonine-protein kinase; ILK-1; ILK-2; p59ILK
Protein Family:	Integrin-linked protein kinase
UniProt Gene Name:	ILK
UniProt Entry Name:	ILK_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-ILK 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.)