Cell Death

TGF beta Receptor II Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB00881
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Death
Reactivity:
Human
Reactivity:
Mouse
Detection Method:
Colorimetric

Description

system_update_altDatasheet
Product Name:TGF beta Receptor II Colorimetric Cell-Based ELISA
Product Code:CBCAB00881
ELISA Type:Cell-Based
Target:TGF beta Receptor II
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The TGF beta Receptor II Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TGF beta Receptor II protein expression profile in cells. The kit can be used for measuring the relative amounts of TGF beta Receptor II in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TGF beta Receptor II.

Qualitative determination of TGF beta Receptor II concentration is achieved by an indirect ELISA format. In essence, TGF beta Receptor II is captured by TGF beta Receptor II-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 7048, UniProt ID: P37173, OMIM: 133239/190182/610168/610380, Unigene: Hs.604277/Hs.82028
Gene Symbol:TGFBR2
Sub Type:None
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
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

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

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies (Anti-TGF beta Receptor II Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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