Paxillin (Phospho-Tyr31)Colorimetric Cell-Based ELISA Kit (CBCAB01595)

Product Name:	Paxillin (Phospho-Tyr31)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01595
ELISA Type:	Cell-Based
Target:	Paxillin (Phospho-Tyr31)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Paxillin (Phospho-Tyr31) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Paxillin protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Paxillin in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Paxillin phosphorylation.

Qualitative determination of Paxillin (Phospho-Tyr31) concentration is achieved by an indirect ELISA format. In essence, Paxillin (Phospho-Tyr31) is captured by Paxillin (Phospho-Tyr31)-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: 5829, UniProt ID: P49023, OMIM: 602505, Unigene: Hs.446336
Gene Symbol:	PXN
Sub Type:	Phospho

UniProt Protein Function:	PXN: a multi-domain cytoskeletal protein involved in actin-membrane attachment that localizes primarily to focal adhesion sites to the extracellular matrix. Phosphorylated by focal adhesion kinase (FAK) and is a component of integrin signaling. Its phosphorylation provides docking sites for recruitment of signaling molecules to focal adhesions. Binds in vitro to vinculin as well as to the SH3 domain of c-SRC and, when tyrosine phosphorylated, to the Crk SH2 domain. Interacts with GIT1, NUDT16L1/SDOS, PARVA and TGFB1I1. Component of cytoplasmic complexes, which also contain GIT1, ARHGEF6 and PAK1. Binds DDEF2. Interacts with unphosphorylated ITGA4. Interacts with RNF5.Three alternatively-spliced isoforms have been described. Isoform beta binds to focal adhesion kinase but weakly to vinculin. Isoform gamma binds to vinculin but only weakly to focal adhesion kinase.
UniProt Protein Details:	Protein type:Adaptor/scaffold; Cytoskeletal; Motility/polarity/chemotaxis; Cell adhesion Chromosomal Location of Human Ortholog: 12q24.31 Cellular Component: nucleoplasm; microtubule associated complex; focal adhesion; lamellipodium; cytoplasm; stress fiber; plasma membrane; cell cortex; cytosol Molecular Function:integrin binding; protein binding; zinc ion binding; beta-catenin binding; BH4 domain binding; protein kinase binding; vinculin binding Biological Process: integrin-mediated signaling pathway; lamellipodium biogenesis; epidermal growth factor receptor signaling pathway; focal adhesion formation; regulation of cell shape; peptidyl-tyrosine phosphorylation; branching morphogenesis of a tube; muscle contraction; activation of MAPK activity; transforming growth factor beta receptor signaling pathway; signal complex assembly; cytoskeleton organization and biogenesis; vascular endothelial growth factor receptor signaling pathway; signal transduction; cell adhesion
NCBI Summary:	This gene encodes a cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix (focal adhesion). Alternatively spliced transcript variants encoding different isoforms have been described for this gene. These isoforms exhibit different expression pattern, and have different biochemical, as well as physiological properties (PMID:9054445). [provided by RefSeq, Aug 2011]
UniProt Code:	P49023
NCBI GenInfo Identifier:	317373486
NCBI Gene ID:	5829
NCBI Accession:	P49023.3
UniProt Secondary Accession:	P49023,O14970, O14971, O60360, Q5HYA4, B2RAI3, B7ZMB4
UniProt Related Accession:	P49023
Molecular Weight:	68kDa
NCBI Full Name:	Paxillin
NCBI Synonym Full Names:	paxillin
NCBI Official Symbol:	PXN
NCBI Protein Information:	paxillin
UniProt Protein Name:	Paxillin
Protein Family:	Paxillin
UniProt Gene Name:	PXN
UniProt Entry Name:	PAXI_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-Paxillin (Phospho-Tyr31) Antibody, Anti-Paxillin 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.)