Human TGFBR2/TGF-beta Receptor II ELISA Kit

Product Name:	Human TGFBR2 / TGF-beta Receptor II ELISA Kit
Product Code:	HUFI00247
Size:	96 Assays
Alias:	TGFBR2, TGF-beta receptor type-2, TGFR-2, AAT3, FAA3, HNPCC6, LDS1B, LDS2B, MFS2, RIIC, TAAD2, TbetaR-II, TGF-beta receptor type II, TGF-beta receptor type IIB, TGF-beta receptor type-2, TGF-beta type II receptor, TGFbeta-RII, TGFR-2
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human TGFBR2 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 TGFBR2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human TGFBR2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-102	95
EDTA plasma(n=5)	89-105	95
UFH plasma(n=5)	88-97	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TGFBR2 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)	93-105%	88-105%	92-101%
EDTA plasma(n=5)	84-100%	83-95%	85-101%
UFH plasma(n=5)	83-95%	87-99%	84-98%

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

P37173

UniProt Protein Function:	TGFBR2: a TKL kinase of the serine/threonine-protein kinase receptor (STKR) family. R1 and R2 TGF-beta receptors dimerize after binding TGF-beta at the cell surface. Binds to DAXX. Defects can cause esophageal cancer.
UniProt Protein Details:	Protein type:Oncoprotein; Membrane protein, integral; Protein kinase, TKL; EC 2.7.11.30; Protein kinase, Ser/Thr (receptor); Kinase, protein; TKL group; STKR family; Type2 subfamily Chromosomal Location of Human Ortholog: 3p22 Cellular Component: caveola; cytosol; external side of plasma membrane; integral to membrane; lipid raft; plasma membrane; receptor complex Molecular Function:ATP binding; glycosaminoglycan binding; metal ion binding; mitogen-activated protein kinase kinase kinase binding; protein binding; receptor signaling protein serine/threonine kinase activity; SMAD binding; transforming growth factor beta binding; transforming growth factor beta receptor activity; transforming growth factor beta receptor activity, type II; transmembrane receptor protein serine/threonine kinase activity Biological Process: activation of protein kinase activity; aging; apoptosis; blood vessel development; brain development; common-partner SMAD protein phosphorylation; embryo implantation; embryonic cranial skeleton morphogenesis; embryonic hemopoiesis; gastrulation; gut development; heart development; in utero embryonic development; lens development in camera-type eye; myeloid dendritic cell differentiation; negative regulation of cardiac muscle cell proliferation; negative regulation of transforming growth factor beta receptor signaling pathway; Notch signaling pathway; organ regeneration; palate development; patterning of blood vessels; peptidyl-serine phosphorylation; peptidyl-threonine phosphorylation; positive regulation of angiogenesis; positive regulation of B cell tolerance induction; positive regulation of cell proliferation; positive regulation of mesenchymal cell proliferation; positive regulation of NK T cell differentiation; positive regulation of skeletal muscle regeneration; positive regulation of smooth muscle cell proliferation; positive regulation of T cell tolerance induction; positive regulation of tolerance induction to self antigen; protein amino acid phosphorylation; receptor-mediated endocytosis; regulation of cell proliferation; regulation of gene expression; response to drug; response to estrogen stimulus; response to glucose stimulus; response to mechanical stimulus; response to nutrient; smoothened signaling pathway; transforming growth factor beta receptor signaling pathway; vasculogenesis; wound healing Disease: Colorectal Cancer, Hereditary Nonpolyposis, Type 6; Esophageal Cancer; Loeys-dietz Syndrome 2
NCBI Summary:	This gene encodes a member of the Ser/Thr protein kinase family and the TGFB receptor subfamily. The encoded protein is a transmembrane protein that has a protein kinase domain, forms a heterodimeric complex with another receptor protein, and binds TGF-beta. This receptor/ligand complex phosphorylates proteins, which then enter the nucleus and regulate the transcription of a subset of genes related to cell proliferation. Mutations in this gene have been associated with Marfan Syndrome, Loeys-Deitz Aortic Aneurysm Syndrome, and the development of various types of tumors. Alternatively spliced transcript variants encoding different isoforms have been characterized. [provided by RefSeq, Jul 2008]
UniProt Code:	P37173
NCBI GenInfo Identifier:	116242818
NCBI Gene ID:	7048
NCBI Accession:	P37173.2
UniProt Secondary Accession:	P37173,Q15580, Q6DKT6, Q99474, B4DTV5,
UniProt Related Accession:	P37173
Molecular Weight:	67,457 Da
NCBI Full Name:	TGF-beta receptor type-2
NCBI Synonym Full Names:	transforming growth factor beta receptor II
NCBI Official Symbol:	TGFBR2
NCBI Official Synonym Symbols:	AAT3; FAA3; LDS2; MFS2; RIIC; LDS1B; LDS2B; TAAD2; TGFR-2; TGFbeta-RII
NCBI Protein Information:	TGF-beta receptor type-2
UniProt Protein Name:	TGF-beta receptor type-2
UniProt Synonym Protein Names:	TGF-beta type II receptor; Transforming growth factor-beta receptor type II; TGF-beta receptor type II; TbetaR-II
Protein Family:	TGF-beta receptor
UniProt Gene Name:	TGFBR2
UniProt Entry Name:	TGFR2_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.