Mouse TGF-beta receptor type-1 (Tgfbr1) ELISA Kit (MOEB2301)

Product Name:	Mouse TGF-beta receptor type-1 (Tgfbr1) ELISA Kit
SKU:	MOEB2301
Size:	96T
Target:	Mouse TGF-beta receptor type-1 (Tgfbr1)
Synonyms:	ESK2, Transforming growth factor-beta receptor type I, TGF-beta receptor type I, TGFR-1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse
Detection Range:	78-5000pg/mL
Sensitivity:	39.4pg/mL

Intra CV:

0.0%

Inter CV:

0.0%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	85-97%	109-119%	105-117%	105-115%
EDTA Plasma(N=5)	102-111%	107-117%	90-99%	107-117%
Heparin Plasma(N=5)	105-105%	94-104%	96-106%	92-104%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	80	80-80
Plasma	80	80-80

Function:

Transmembrane serine/threonine kinase forming with the TGF-beta type II serine/threonine kinase receptor, TGFBR2, the non-promiscuous receptor for the TGF-beta cytokines TGFB1, TGFB2 and TGFB3. Transduces the TGFB1, TGFB2 and TGFB3 signal from the cell surface to the cytoplasm and is thus regulating a plethora of physiological and pathological processes including cell cycle arrest in epithelial and hematopoietic cells, control of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression and carcinogenesis. The formation of the receptor complex composed of 2 TGFBR1 and 2 TGFBR2 molecules symmetrically bound to the cytokine dimer results in the phosphorylation and the activation of TGFBR1 by the constitutively active TGFBR2. Activated TGFBR1 phosphorylates SMAD2 which dissociates from the receptor and interacts with SMAD4. The SMAD2-SMAD4 complex is subsequently translocated to the nucleus where it modulates the transcription of the TGF-beta-regulated genes. This constitutes the canonical SMAD-dependent TGF-beta signaling cascade. Also involved in non-canonical, SMAD-independent TGF-beta signaling pathways. For instance, TGFBR1 induces TRAF6 autoubiquitination which in turn results in MAP3K7 ubiquitination and activation to trigger apoptosis. Also regulates epithelial to mesenchymal transition through a SMAD-independent signaling pathway through PARD6A phosphorylation and activation.

Uniprot:	Q64729
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse TGF-beta receptor type-1
Sub Unit:	Homodimer; in the endoplasmic reticulum but also at the cell membrane. Heterohexamer; TGFB1, TGFB2 and TGFB3 homodimeric ligands assemble a functional receptor composed of two TGFBR1 and TGFBR2 heterodimers to form a ligand-receptor heterohexamer. The respective affinity of TGBRB1 and TGFBR2 for the ligands may modulate the kinetics of assembly of the receptor and may explain the different biological activities of TGFB1, TGFB2 and TGFB3. Interacts with CD109; inhibits TGF-beta receptor activation in keratinocytes. Interacts with RBPMS. Interacts (unphosphorylated) with FKBP1A; prevents TGFBR1 phosphorylation by TGFBR2 and stabilizes it in the inactive conformation. Interacts with SMAD2, SMAD3 and ZFYVE9; ZFYVE9 recruits SMAD2 and SMAD3 to the TGF-beta receptor. Interacts with TRAF6 and MAP3K7; induces MAP3K7 activation by TRAF6. Interacts with PARD6A; involved in TGF-beta induced epithelial to mesenchymal transition. Interacts with SMAD7, NEDD4L, SMURF1 and SMURF2; SMAD7 recruits NEDD4L, SMURF1 and SMURF2 to the TGF-beta receptor (By similarity). Interacts with USP15 and VPS39. Interacts with SDCBP (via C-terminus). Interacts with CAV1 and this interaction is impaired in the presence of SDCBP.
Research Area:	Cancer
Subcellular Location:	Cell membrane Single-pass type I membrane protein Cell junction Tight junction Membrane raft Cell surface
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	TGFBR1: 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. Found in all tissues; most abundant in placenta and least abundant in brain and heart.Protein type: EC 2.7.11.30; Protein kinase, Ser/Thr (receptor); Receptor, misc.; Kinase, protein; Membrane protein, integral; Protein kinase, TKL; TKL group; STKR family; Type1 subfamilyCellular Component: apical plasma membrane; basolateral plasma membrane; caveola; cell surface; endosome; lipid raft; plasma membrane; protein complex; receptor complex; tight junctionMolecular Function: ATP binding; growth factor binding; protein binding; protein complex binding; protein heterodimerization activity; protein kinase activity; protein serine/threonine kinase activity; punt binding; receptor 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 I; ubiquitin protein ligase bindingBiological Process: activation of MAPKK activity; angiogenesis; anterior/posterior pattern formation; artery morphogenesis; blastocyst development; cell motility involved in cell locomotion; collagen fibril organization; embryonic cranial skeleton morphogenesis; embryonic development; endothelial cell migration; epithelial to mesenchymal transition; germ cell migration; heart development; in utero embryonic development; kidney development; lens development in camera-type eye; male gonad development; negative regulation of apoptosis; negative regulation of chondrocyte differentiation; negative regulation of endothelial cell differentiation; negative regulation of endothelial cell proliferation; neuron fate commitment; palate development; parathyroid gland development; peptidyl-serine phosphorylation; peptidyl-threonine phosphorylation; pharyngeal system development; positive regulation of cell growth; positive regulation of cell migration; positive regulation of cell motility; positive regulation of cell proliferation; positive regulation of filopodium formation; positive regulation of protein kinase B signaling cascade; positive regulation of transcription, DNA-dependent; post-embryonic development; protein amino acid autophosphorylation; protein amino acid phosphorylation; regulation of gene expression; regulation of protein binding; regulation of protein ubiquitination; regulation of transcription, DNA-dependent; response to estrogen stimulus; signal transduction; skeletal development; skeletal morphogenesis; thymus development; transforming growth factor beta receptor signaling pathway
UniProt Protein Details:
NCBI Summary:	This gene encodes a member of the transforming growth factor beta (TGF-beta) receptor family of proteins. These proteins comprise one component of the TGF-beta signaling pathway, which transduces extracellular signals into gene expression changes to regulate a wide range of cellular responses, including proliferation, migration, differentiation and apoptosis. Homozygous knockout mice for this gene exhibit impaired angiogenesis and embryonic lethality. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Aug 2015]
UniProt Code:	Q64729
NCBI GenInfo Identifier:	6094468
NCBI Gene ID:	21812
NCBI Accession:	Q64729.1
UniProt Secondary Accession:	Q64729,A2AJN0
UniProt Related Accession:	Q64729
Molecular Weight:	55,790 Da
NCBI Full Name:	TGF-beta receptor type-1
NCBI Synonym Full Names:	transforming growth factor, beta receptor I
NCBI Official Symbol:	Tgfbr1
NCBI Official Synonym Symbols:	ALK5; ESK2; Alk-5; TGFR-1; TbetaRI; AU017191; TbetaR-I
NCBI Protein Information:	TGF-beta receptor type-1
UniProt Protein Name:	TGF-beta receptor type-1
UniProt Synonym Protein Names:	ESK2; Transforming growth factor-beta receptor type I; TGF-beta receptor type I; TbetaR-I
Protein Family:	TGF-beta receptor
UniProt Gene Name:	Tgfbr1
UniProt Entry Name:	TGFR1_MOUSE

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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 TGF-beta receptor type-1 (Tgfbr1) ELISA Kit (MOEB2301)

Description