ICAM-1 (Phospho-Tyr512)Colorimetric Cell-Based ELISA Kit (CBCAB01547)

Product Name:	ICAM-1 (Phospho-Tyr512)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01547
ELISA Type:	Cell-Based
Target:	ICAM-1 (Phospho-Tyr512)
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The ICAM-1 (Phospho-Tyr512) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ICAM-1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ICAM-1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on ICAM-1 phosphorylation.

Qualitative determination of ICAM-1 (Phospho-Tyr512) concentration is achieved by an indirect ELISA format. In essence, ICAM-1 (Phospho-Tyr512) is captured by ICAM-1 (Phospho-Tyr512)-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: 3383, UniProt ID: P05362, OMIM: 147840, Unigene: Hs.706750/Hs.707983
Gene Symbol:	ICAM1
Sub Type:	Phospho

UniProt Protein Function:	ICAM1: a type I membrane protein of the immunoglobulin superfamily. Is a ligand for the leukocyte adhesion LFA-1 protein (Integrin alpha-L/beta-2) and a Rhinovirus receptor. Typically expressed on endothelial cells and cells of the immune system. ICAM1 binds to integrins of type CD11a / CD18, or CD11b / CD18. Its expression is activated by p53 in an NF-kappaB-independent manner. Induced by TNFalpha in a process that involves IKKbeta.
UniProt Protein Details:	Protein type:Immunoglobulin superfamily; Membrane protein, integral; Cell adhesion Chromosomal Location of Human Ortholog: 19p13.3-p13.2 Cellular Component: extracellular space; focal adhesion; cell surface; membrane; integral to plasma membrane; plasma membrane; immunological synapse; external side of plasma membrane Molecular Function:viral receptor activity; integrin binding; protein binding; transmembrane receptor activity; receptor activity Biological Process: entry of virus into host cell; extracellular matrix organization and biogenesis; positive regulation of nitric oxide biosynthetic process; T cell antigen processing and presentation; response to organic cyclic substance; activation of NF-kappaB transcription factor; positive regulation of cellular extravasation; regulation of cell shape; leukocyte adhesion; cellular response to nutrient levels; sensory perception of sound; ovarian follicle development; T cell activation via T cell receptor contact with antigen bound to MHC molecule on antigen presenting cell; membrane to membrane docking; response to sulfur dioxide; cell adhesion; regulation of leukocyte mediated cytotoxicity; acute inflammatory response to antigenic stimulus; response to drug; regulation of cell adhesion; virion attachment, binding of host cell surface receptor; negative regulation of calcium ion transport; regulation of immune response; cytokine and chemokine mediated signaling pathway; response to amphetamine; cell aging; response to amino acid stimulus; heterophilic cell adhesion; positive regulation of peptidyl-tyrosine phosphorylation; response to ethanol; response to copper ion; positive regulation of actin filament polymerization; positive regulation of vasoconstriction; adhesion to host; cell adhesion mediated by integrin; response to ionizing radiation; leukocyte migration Disease: Malaria, Susceptibility To
NCBI Summary:	This gene encodes a cell surface glycoprotein which is typically expressed on endothelial cells and cells of the immune system. It binds to integrins of type CD11a / CD18, or CD11b / CD18 and is also exploited by Rhinovirus as a receptor. [provided by RefSeq, Jul 2008]
UniProt Code:	P05362
NCBI GenInfo Identifier:	68067956
NCBI Gene ID:	3383
NCBI Accession:	P05362.2
UniProt Secondary Accession:	P05362,Q5NKV7, Q96B50, B2R6M3,
UniProt Related Accession:	P05362
Molecular Weight:
NCBI Full Name:	Intercellular adhesion molecule 1
NCBI Synonym Full Names:	intercellular adhesion molecule 1
NCBI Official Symbol:	ICAM1
NCBI Official Synonym Symbols:	BB2; CD54; P3.58
NCBI Protein Information:	intercellular adhesion molecule 1; ICAM-1; cell surface glycoprotein P3.58; major group rhinovirus receptor; intercellular adhesion molecule 1 (CD54), human rhinovirus receptor
UniProt Protein Name:	Intercellular adhesion molecule 1
UniProt Synonym Protein Names:	Major group rhinovirus receptor; CD_antigen: CD54
Protein Family:	Intercellular adhesion molecule
UniProt Gene Name:	ICAM1
UniProt Entry Name:	ICAM1_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-ICAM-1 (Phospho-Tyr512) Antibody, Anti-ICAM-1 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.)