Product Name:	Human FAK / Focal adhesion kinase 1 ELISA Kit
Product Code:	HUFI00420
Size:	96 Assays
Alias:	PTK2, FAK, FADK1, FADK 1, FADK, FAK-related non-kinase polypeptide, FAK1, FRNK, focal adhesion kinase 1, pp125FAK, Protein-tyrosine kinase 2, p125FAK, PPP1R71
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PTK2 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.938ng/ml
Range:	1.563-100ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human PTK2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human PTK2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	91-104	99
EDTA plasma(n=5)	87-104	98
UFH plasma(n=5)	89-93	91

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PTK2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	86-105%	90-103%	91-100%
EDTA plasma(n=5)	83-98%	82-97%	83-94%
UFH plasma(n=5)	80-100%	84-99%	81-96%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

Q05397

UniProt Protein Function:	FAK: a tyrosine kinase of the FAK family required for cell migration and contact-dependent survival signaling. Activated by tyrosine-phosphorylation in response to either integrin clustering induced by cell adhesion or antibody cross-linking, or via G-protein coupled receptor (GPCR) occupancy by ligands such as bombesin or lysophosphatidic acid, or via LDL receptor occupancy. Downstream of integrins and Src, upstream of Ras/MAPK. Localizes to focal adhesions that form between cells growing in the presence of extracellular matrix constituents. Interacts with CAS family members and with GIT1, SORBS1 and BCAR3. Interacts with Shb. Required for full Ras transformation of fibroblasts. Increased expression in breast and other cancers, related to chromosome 8q amplification. Overexpression and activation associated with increased migration, invasion and progression of ovarian cancer, and with progression in hepatocellular carcinoma, thyroid cancer, and acute myelogenous leukemia. siRNA increases chemosensitivity of pancreatic adenocarcinoma xenografts. Inhibitor: ISI15421 (antisense). Four splice-variant isoforms have been observed.
UniProt Protein Details:	Protein type:Kinase, protein; Protein kinase, tyrosine (non-receptor); EC 2.7.10.2; Protein kinase, TK; TK group; Fak family Chromosomal Location of Human Ortholog: 8q24.3 Cellular Component: extrinsic to internal side of plasma membrane; cytoskeleton; focal adhesion; lamellipodium; apical plasma membrane; cytoplasm; stress fiber; plasma membrane; microtubule organizing center; cell cortex; cytosol; nucleus Molecular Function:JUN kinase binding; signal transducer activity; protein binding; protein-tyrosine kinase activity; non-membrane spanning protein tyrosine kinase activity; SH2 domain binding; actin binding; protein kinase binding; ATP binding; protein kinase activity; receptor binding Biological Process: heart morphogenesis; axon guidance; extracellular matrix organization and biogenesis; peptidyl-tyrosine phosphorylation; establishment of nucleus localization; apoptosis; protein amino acid autophosphorylation; neuron migration; cell motility involved in cell locomotion; negative regulation of synaptogenesis; regulation of cell shape; regulation of cell adhesion mediated by integrin; transforming growth factor beta receptor signaling pathway; positive regulation of cell proliferation; ephrin receptor signaling pathway; negative regulation of axonogenesis; angiogenesis; vasculogenesis; placenta development; cell structure disassembly during apoptosis; integrin-mediated signaling pathway; epidermal growth factor receptor signaling pathway; platelet activation; central nervous system neuron axonogenesis; regulation of osteoblast differentiation; positive regulation of phosphoinositide 3-kinase activity; signal complex assembly; cytoskeleton organization and biogenesis; microtubule cytoskeleton organization and biogenesis; negative regulation of organ growth; regulation of cell proliferation; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of protein kinase B signaling cascade; embryonic development; establishment of cell polarity; positive regulation of protein kinase activity; regulation of focal adhesion formation; endothelial cell migration; innate immune response; positive regulation of protein amino acid phosphorylation; negative regulation of cell-cell adhesion; blood coagulation; vascular endothelial growth factor receptor signaling pathway; regulation of cytoskeleton organization and biogenesis; negative regulation of apoptosis; positive regulation of cell migration
NCBI Summary:	This gene encodes a cytoplasmic protein tyrosine kinase which is found concentrated in the focal adhesions that form between cells growing in the presence of extracellular matrix constituents. The encoded protein is a member of the FAK subfamily of protein tyrosine kinases but lacks significant sequence similarity to kinases from other subfamilies. Activation of this gene may be an important early step in cell growth and intracellular signal transduction pathways triggered in response to certain neural peptides or to cell interactions with the extracellular matrix. Several transcript variants encoding different isoforms have been found for this gene, but the full-length natures of only three of them have been determined. [provided by RefSeq, Dec 2010]
UniProt Code:	Q05397
NCBI GenInfo Identifier:	3183518
NCBI Gene ID:	5747
NCBI Accession:	Q05397.2
UniProt Secondary Accession:	Q05397,Q14291, Q8IYN9, Q9UD85, B4E2N6, F5H4S4, J3QT16
UniProt Related Accession:	Q05397
Molecular Weight:	1052
NCBI Full Name:	Focal adhesion kinase 1
NCBI Synonym Full Names:	protein tyrosine kinase 2
NCBI Official Symbol:	PTK2
NCBI Official Synonym Symbols:	FAK; FADK; FAK1; FRNK; PPP1R71; p125FAK; pp125FAK
NCBI Protein Information:	focal adhesion kinase 1; FADK 1; PTK2 protein tyrosine kinase 2; FAK-related non-kinase polypeptide; focal adhesion kinase isoform FAK-Del33; focal adhesion kinase-related nonkinase; protein phosphatase 1 regulatory subunit 71; protein phosphatase 1, regulatory subunit 71
UniProt Protein Name:	Focal adhesion kinase 1
UniProt Synonym Protein Names:	Focal adhesion kinase-related nonkinase; FRNK; Protein phosphatase 1 regulatory subunit 71; PPP1R71; Protein-tyrosine kinase 2; p125FAK; pp125FAK
Protein Family:	Focal adhesion kinase
UniProt Gene Name:	PTK2
UniProt Entry Name:	FAK1_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.