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TNFSF9 Colorimetric Cell-Based ELISA

SKU:
CBCAB01013
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Detection Method:
Colorimetric
$719
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Description

system_update_altDatasheetMSDS

TNFSF9 Colorimetric Cell-Based ELISA

The TNFSF9 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for accurately measuring levels of TNFSF9 (tumor necrosis factor superfamily 9) in cell culture supernatants. This kit is designed with high sensitivity and specificity, ensuring reliable and reproducible results for a variety of research applications.TNFSF9 is a key protein involved in immune response and cell signaling, playing a critical role in various diseases such as cancer, autoimmune disorders, and inflammatory conditions.

Studying TNFSF9 levels can provide valuable insights into the mechanisms underlying these diseases and aid in the development of targeted therapies.With the TNFSF9 Colorimetric Cell-Based ELISA Kit, researchers can easily and efficiently measure TNFSF9 levels in cell cultures, leading to a better understanding of disease processes and the potential for the discovery of novel therapeutic strategies.

Product Name:TNFSF9 Colorimetric Cell-Based ELISA
Product Code:CBCAB01013
ELISA Type:Cell-Based
Target:TNFSF9
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The TNFSF9 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TNFSF9 protein expression profile in cells. The kit can be used for measuring the relative amounts of TNFSF9 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TNFSF9.

Qualitative determination of TNFSF9 concentration is achieved by an indirect ELISA format. In essence, TNFSF9 is captured by TNFSF9-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: 8744, UniProt ID: P41273, OMIM: 606182, Unigene: Hs.1524
Gene Symbol:TNFSF9
Sub Type:None
UniProt Protein Function:TNFL9: Cytokine that binds to TNFRSF9. Induces the proliferation of activated peripheral blood T-cells. May have a role in activation-induced cell death (AICD). May play a role in cognate interactions between T-cells and B-cells/macrophages. Belongs to the tumor necrosis factor family.
UniProt Protein Details:

Protein type:Apoptosis; Cell cycle regulation; Cytokine; Membrane protein, integral

Chromosomal Location of Human Ortholog: 19p13.3

Cellular Component: plasma membrane

Molecular Function:receptor binding; tumor necrosis factor receptor superfamily binding

Biological Process: apoptosis; cell proliferation; cell-cell signaling; positive regulation of activated T cell proliferation; positive regulation of cytotoxic T cell differentiation; signal transduction; tumor necrosis factor-mediated signaling pathway

NCBI Summary:The protein encoded by this gene is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This transmembrane cytokine is a bidirectional signal transducer that acts as a ligand for TNFRSF9/4-1BB, which is a costimulatory receptor molecule in T lymphocytes. This cytokine and its receptor are involved in the antigen presentation process and in the generation of cytotoxic T cells. The receptor TNFRSF9/4-1BB is absent from resting T lymphocytes but rapidly expressed upon antigenic stimulation. The ligand encoded by this gene, TNFSF9/4-1BBL, has been shown to reactivate anergic T lymphocytes in addition to promoting T lymphocyte proliferation. This cytokine has also been shown to be required for the optimal CD8 responses in CD8 T cells. This cytokine is expressed in carcinoma cell lines, and is thought to be involved in T cell-tumor cell interaction.[provided by RefSeq, Oct 2008]
UniProt Code:P41273
NCBI GenInfo Identifier:728739
NCBI Gene ID:8744
NCBI Accession:P41273.1
UniProt Secondary Accession:P41273,Q2M3S2,
UniProt Related Accession:P41273
Molecular Weight:27kDa
NCBI Full Name:Tumor necrosis factor ligand superfamily member 9
NCBI Synonym Full Names:TNF superfamily member 9
NCBI Official Symbol:TNFSF9  
NCBI Official Synonym Symbols:CD137L; TNLG5A; 4-1BB-L  
NCBI Protein Information:tumor necrosis factor ligand superfamily member 9
UniProt Protein Name:Tumor necrosis factor ligand superfamily member 9
UniProt Synonym Protein Names:4-1BB ligand; 4-1BBL
Protein Family:Tumor necrosis factor ligand superfamily
UniProt Gene Name:TNFSF9  
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-TNFSF9 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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