Human PTEN ELISA Kit (HUFI01372)- High Sensitivity

Product Name:	Human PTEN ELISA Kit
Product Code:	HUFI01372
Size:	96 Assays
Alias:	PTEN, Phosphatidylinositol-3, 4, 5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN, Mutated in multiple advanced cancers 1, Phosphatase and tensin homolog, MMAC1, TEP1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human PTEN concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

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

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-101	95
EDTA plasma(n=5)	89-103	98
UFH plasma(n=5)	85-102	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PTEN 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)	90-105%	86-105%	92-105%
EDTA plasma(n=5)	84-98%	87-94%	84-101%
UFH plasma(n=5)	86-93%	85-96%	86-100%

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

P60484

UniProt Protein Function:	PTEN: a phosphoinositide 3-phosphatase (PIP3) and a tumor suppressor implicated in a wide variety of human cancers. Dephosphorylates inositol phospholipids generated by the activation of the phosphoinositide 3 kinase (PI3K). A major negative regulator of the PI3K/Akt signaling pathway. Possesses a C-terminal regulatory domain that contains three phosphorylation sites which may regulate its stability.
UniProt Protein Details:	Protein type:Carbohydrate Metabolism - inositol phosphate; EC 3.1.3.48; Phosphatase, lipid; Nuclear receptor co-regulator; Motility/polarity/chemotaxis; EC 3.1.3.16; EC 3.1.3.67; Tumor suppressor Chromosomal Location of Human Ortholog: 10q23.3 Cellular Component: nucleoplasm; postsynaptic membrane; PML body; internal side of plasma membrane; neuron projection; cell projection; mitochondrion; cytoplasm; plasma membrane; extracellular region; dendritic spine; cytosol; nucleus Molecular Function:phosphatidylinositol-3-phosphatase activity; phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase activity; inositol-1,3,4,5-tetrakisphosphate 3-phosphatase activity; platelet-derived growth factor receptor binding; magnesium ion binding; protein tyrosine phosphatase activity; protein serine/threonine phosphatase activity; protein kinase binding; PDZ domain binding; protein binding; enzyme binding; phosphatidylinositol-3,4-bisphosphate 3-phosphatase activity; protein tyrosine/serine/threonine phosphatase activity; phosphoprotein phosphatase activity; lipid binding Biological Process: central nervous system development; male mating behavior; nerve growth factor receptor signaling pathway; prepulse inhibition; response to arsenic; regulation of cellular component size; heart development; anaphase-promoting complex activation during mitotic cell cycle; Wnt receptor signaling pathway through beta-catenin; T cell receptor signaling pathway; regulation of B cell apoptosis; myelin maintenance in the central nervous system; response to glucose stimulus; negative regulation of axonogenesis; response to drug; rhythmic synaptic transmission; fibroblast growth factor receptor signaling pathway; dentate gyrus development; negative regulation of myelination; social behavior; forebrain morphogenesis; negative regulation of organ growth; response to ethanol; response to zinc ion; inositol phosphate dephosphorylation; phosphatidylinositol biosynthetic process; endothelial cell migration; positive regulation of transcription factor activity; multicellular organismal response to stress; negative regulation of apoptosis; negative regulation of phagocytosis; synapse maturation; inositol phosphate metabolic process; apoptosis; regulation of protein stability; locomotory behavior; negative regulation of cell size; platelet-derived growth factor receptor signaling pathway; protein amino acid dephosphorylation; regulation of myeloid cell apoptosis; response to estradiol stimulus; negative regulation of cell proliferation; locomotor rhythm; learning and/or memory; synaptogenesis; negative regulation of protein amino acid phosphorylation; phospholipid metabolic process; positive regulation of cell proliferation; brain morphogenesis; protein kinase B signaling cascade; nerve-nerve synaptic transmission; angiogenesis; negative regulation of cell migration; response to nutrient; negative regulation of epithelial cell proliferation; aging; cardiac muscle development; epidermal growth factor receptor signaling pathway; cell migration; phosphoinositide-mediated signaling; negative regulation of protein kinase B signaling cascade; protein stabilization; central nervous system neuron axonogenesis; maternal behavior; negative regulation of focal adhesion formation; response to ATP; memory; cell proliferation; G1/S-specific negative regulation of cyclin-dependent protein kinase activity; innate immune response; phosphoinositide dephosphorylation; regulation of cyclin-dependent protein kinase activity; negative regulation of phosphoinositide 3-kinase cascade Disease: Meningioma, Familial, Susceptibility To; Thyroid Carcinoma, Follicular; Melanoma, Cutaneous Malignant, Susceptibility To, 1; Bannayan-riley-ruvalcaba Syndrome; Cowden Syndrome 1; Squamous Cell Carcinoma, Head And Neck; Prostate Cancer; Glioma Susceptibility 2; Vacterl Association With Hydrocephalus; Endometrial Cancer; Macrocephaly/autism Syndrome
NCBI Summary:	This gene was identified as a tumor suppressor that is mutated in a large number of cancers at high frequency. The protein encoded this gene is a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. It contains a tensin like domain as well as a catalytic domain similar to that of the dual specificity protein tyrosine phosphatases. Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates. It negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating AKT/PKB signaling pathway. [provided by RefSeq, Jul 2008]
UniProt Code:	P60484
NCBI GenInfo Identifier:	42560209
NCBI Gene ID:	5728
NCBI Accession:	P60484.1
UniProt Secondary Accession:	P60484,O00633, O02679, Q6ICT7, B2R904, F2YHV0,
UniProt Related Accession:	P60484
Molecular Weight:	19,796 Da
NCBI Full Name:	Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN
NCBI Synonym Full Names:	phosphatase and tensin homolog
NCBI Official Symbol:	PTEN
NCBI Official Synonym Symbols:	BZS; DEC; CWS1; GLM2; MHAM; TEP1; MMAC1; PTEN1; 10q23del
NCBI Protein Information:	phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN; mitochondrial PTENalpha; phosphatase and tensin-like protein; mutated in multiple advanced cancers 1; mitochondrial phosphatase and tensin protein alpha; MMAC1 phosphatase and tensin homolog deleted on chromosome 10; phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN
UniProt Protein Name:	Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN
UniProt Synonym Protein Names:	Mutated in multiple advanced cancers 1; Phosphatase and tensin homolog
Protein Family:	Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase
UniProt Gene Name:	PTEN
UniProt Entry Name:	PTEN_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.