Mouse TEK/TIE2 ELISA Kit (MOFI00169)

Product Name:	Mouse TEK/TIE2 ELISA Kit
Product Code:	MOFI00169
Size:	96 Assays
Alias:	Tek, Tie-2, TEK, CD202B, TIE2, VMCM, VMCM1, Angiopoietin-1 receptor, ANG-R-Tie2, Angiopoietin Receptor Tie2
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse Tek 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 Mouse Tek and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse Tek in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-105	99
EDTA plasma(n=5)	89-105	95
UFH plasma(n=5)	85-95	91

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse Tek 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)	87-96%	90-105%	86-96%
EDTA plasma(n=5)	85-96%	83-101%	83-97%
UFH plasma(n=5)	86-98%	82-100%	83-96%

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

Q02858

UniProt Protein Function:	TIE2: a receptor tyrosine kinase of the Tie family. Receptor for angiopoietin 1. Expressed almost exclusively in endothelial cells. May constitute the earliest mammalian endothelial cell lineage marker. Appears to be critical for endothelial cell-smooth muscle cell communication in venous morphogenesis. TEK is closely related to the TIE receptor tyrosine kinase.
UniProt Protein Details:	Protein type:Kinase, protein; Membrane protein, integral; EC 2.7.10.1; Protein kinase, tyrosine (receptor); Protein kinase, TK; TK group; Tie family Cellular Component: microvillus; cell surface; integral to plasma membrane; basolateral plasma membrane; extracellular region; integral to membrane; actin filament; intercellular junction; lipid raft; cytoskeleton; membrane; perinuclear region of cytoplasm; apical plasma membrane; cytoplasm; basal plasma membrane; stress fiber; plasma membrane; cell junction Molecular Function:transferase activity; protein binding; protein-tyrosine kinase activity; growth factor binding; transferase activity, transferring phosphorus-containing groups; nucleotide binding; kinase activity; receptor activity; transmembrane receptor protein tyrosine kinase activity; ATP binding; protein kinase activity Biological Process: positive regulation of cell adhesion; peptidyl-tyrosine phosphorylation; heart development; protein amino acid autophosphorylation; cell-matrix adhesion; positive regulation of cytokine secretion during immune response; protein amino acid phosphorylation; regulation of cell migration; positive regulation of vascular endothelial growth factor receptor signaling pathway; cell-cell adhesion; positive regulation of focal adhesion formation; hemopoiesis; angiogenesis; vasculogenesis; Tie receptor signaling pathway; regulation of endothelial cell proliferation; response to retinoic acid; regulation of establishment and/or maintenance of cell polarity; positive regulation of phosphoinositide 3-kinase activity; regulation of angiogenesis; positive regulation of peptidyl-serine phosphorylation; protein oligomerization; positive regulation of phosphoinositide 3-kinase cascade; patterning of blood vessels; positive regulation of protein kinase B signaling cascade; negative regulation of angiogenesis; positive regulation of angiogenesis; positive regulation of protein import into nucleus; response to estrogen stimulus; endothelial cell proliferation; positive regulation of protein amino acid phosphorylation; sprouting angiogenesis; phosphorylation; transmembrane receptor protein tyrosine kinase signaling pathway; negative regulation of apoptosis
UniProt Code:	Q02858
NCBI GenInfo Identifier:	730947
NCBI Gene ID:	21687
NCBI Accession:	Q02858.2
UniProt Related Accession:	Q02858
Molecular Weight:	125,701 Da
NCBI Full Name:	Angiopoietin-1 receptor
NCBI Synonym Full Names:	endothelial-specific receptor tyrosine kinase
NCBI Official Symbol:	Tek
NCBI Official Synonym Symbols:	Hyk; STK1; Tie2; Tie-2; Cd202b; AA517024
NCBI Protein Information:	angiopoietin-1 receptor; p140 TEK; endothelial tyrosine kinase; tyrosine-protein kinase receptor TEK; tunica interna endothelial cell kinase; tyrosine-protein kinase receptor TIE-2; tyrosine kinase with Ig and EGF homology domains-2
UniProt Protein Name:	Angiopoietin-1 receptor
UniProt Synonym Protein Names:	Endothelial tyrosine kinase; HYK; STK1; Tunica interna endothelial cell kinase; Tyrosine kinase with Ig and EGF homology domains-2; Tyrosine-protein kinase receptor TEK; Tyrosine-protein kinase receptor TIE-2; mTIE2; p140 TEK; CD_antigen: CD202b
Protein Family:	Angiopoietin-1 receptor
UniProt Gene Name:	Tek
UniProt Entry Name:	TIE2_MOUSE

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

Step	Procedure
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.

Mouse TEK/TIE2 ELISA Kit (MOFI00169)

Description