TNF Receptor II Colorimetric Cell-Based ELISA

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SKU:
CBCAB00985
€499

Description

Technical Manual

TNF Receptor II Cell-Based ELISA Kit

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

How does our TNF Receptor II Colorimetric Cell-Based ELISA Kit work?

Qualitative determination of TNF Receptor II concentration is achieved by an indirect ELISA format. In essence, TNF Receptor II is captured by TNF Receptor II -specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° 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.

TNF Receptor II Colorimetric Cell-Based ELISA - Information

Product Name:TNF Receptor II Colorimetric Cell-Based ELISA
Product Code:/SKUCBCAB00985
Description:The TNF Receptor II Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TNF Receptor II protein expression profile in cells. The kit can be used for measuring the relative amounts of TNF Receptor II in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TNF Receptor II.
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based
Database Links:Gene ID: 7133, UniProt ID: P20333, OMIM #: 191191, Unigene #: Hs.256278
Format:96-Well Microplate
NCBI Gene Symbol:TNFRSF1B
Sub Type:None
Target Name:TNF Receptor II

Kit Principle

Figure: Schematic representation of ELISA Genie Cell-Based Colorimetric ELISA principle

Kit components

Quantity

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 ul
100x Anti-Target Antibody60 ul
Anti-GAPDH Antibody60 ul
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

Additional equipment and materials required

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

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

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-TNF 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.)

TNF Receptor II - Protein Information

UniProt Protein Function:TNF-R2: Receptor with high affinity for TNFSF2/TNF-alpha and approximately 5-fold lower affinity for homotrimeric TNFSF1/lymphotoxin-alpha. The TRAF1/TRAF2 complex recruits the apoptotic suppressors BIRC2 and BIRC3 to TNFRSF1B/TNFR2. This receptor mediates most of the metabolic effects of TNF-alpha. Isoform 2 blocks TNF-alpha-induced apoptosis, which suggests that it regulates TNF-alpha function by antagonizing its biological activity. Binds to TRAF2. Interacts with BMX. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Membrane protein, integral; Receptor, cytokine

Chromosomal Location of Human Ortholog: 1p36.22

Cellular Component: cell soma; perinuclear region of cytoplasm; integral to membrane; plasma membrane; extracellular region; nucleus; lipid raft

Molecular Function:protein binding; tumor necrosis factor receptor activity; ubiquitin protein ligase binding

Biological Process: tumor necrosis factor-mediated signaling pathway; DNA damage response, signal transduction resulting in induction of apoptosis; negative regulation of inflammatory response; immune response; RNA destabilization; inflammatory response; positive regulation of membrane protein ectodomain proteolysis; aging

NCBI Summary:The protein encoded by this gene is a member of the TNF-receptor superfamily. This protein and TNF-receptor 1 form a heterocomplex that mediates the recruitment of two anti-apoptotic proteins, c-IAP1 and c-IAP2, which possess E3 ubiquitin ligase activity. The function of IAPs in TNF-receptor signalling is unknown, however, c-IAP1 is thought to potentiate TNF-induced apoptosis by the ubiquitination and degradation of TNF-receptor-associated factor 2, which mediates anti-apoptotic signals. Knockout studies in mice also suggest a role of this protein in protecting neurons from apoptosis by stimulating antioxidative pathways. [provided by RefSeq, Jul 2008]
UniProt Code:P20333
NCBI GenInfo Identifier:21264534
NCBI Gene ID:7133
NCBI Accession:P20333.3
UniProt Secondary Accession:P20333,Q16042, Q6YI29, Q9UIH1, B1AJZ3,
UniProt Related Accession:P20333
Molecular Weight:28,461 Da
NCBI Full Name:Tumor necrosis factor receptor superfamily member 1B
NCBI Synonym Full Names:tumor necrosis factor receptor superfamily, member 1B
NCBI Official Symbol:TNFRSF1B  
NCBI Official Synonym Symbols:p75; TBPII; TNFBR; TNFR2; CD120b; TNFR1B; TNFR80; TNF-R75; p75TNFR; TNF-R-II  
NCBI Protein Information:tumor necrosis factor receptor superfamily member 1B; TNF-R2; TNF-RII; p75 TNF receptor; p80 TNF-alpha receptor; soluble TNFR1B variant 1; tumor necrosis factor receptor 2; tumor necrosis factor beta receptor; tumor necrosis factor receptor type II; tumor necrosis factor binding protein 2
UniProt Protein Name:Tumor necrosis factor receptor superfamily member 1B
UniProt Synonym Protein Names:Tumor necrosis factor receptor 2; TNF-R2; Tumor necrosis factor receptor type II; TNF-RII; TNFR-II; p75; p80 TNF-alpha receptor; CD_antigen: CD120b; INN: EtanerceptCleaved into the following 2 chains:Tumor necrosis factor receptor superfamily member 1b, membrane form; Tumor necrosis factor-binding protein 2Alternative name(s):TBP-2; TBPII
UniProt Gene Name:TNFRSF1B  
UniProt Entry Name:TNR1B_HUMAN
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