Human CD178 ELISpot Pair

SKU:
HUDC0161
€670

Description

ELISA Kit Technical ManualMSDS

Human CD178 ELISpot Pair

Assay Genie ELISpot is a highly specific immunoassay for the analysis of CD178 production and secretion from T-cells at a single cell level in conditions closely comparable to the in-vivo environment with minimal cell manipulation. This technique is designed to determine the frequency of CD178 producing cells under a given stimulation and the comparison of such frequency against a specific treatment or pathological state. Utilising sandwich immuno-enzyme technology, Assay Genie ELISpot assays can detect both secreted CD178 (qualitative analysis) and single cells that produce CD178 (quantitative analysis). Cell secreted CD178 is captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured CD178 is revealed by tracer antibodies and appropriate conjugates.

Human CD178 ELISpot Pair - Kit Principle

A capture antibody highly specific for CD178 is coated to the wells of a PVDF bottomed 96 well microtitre plate either during kit manufacture or in the laboratory. The plate is then blocked to minimise any non-antibody dependent unspecific binding and washed. Cell suspension and stimulant are added and the plate incubated allowing the specific antibodies to bind any CD178 produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured CD178. Enzyme conjugated streptavidin is then added binding to the detection antibodies. Following incubation and washing, substrate is then applied to the wells resulting in coloured spots which can be quantified using appropriate analysis software or manually using a microscope.

ELISpot Protocol
Figure: Schematic representation of the principle of ELISpot. 1) Treat with ethanol, coat with capture antibody. 2) Incubate cells in the presence of stimuli, cytokines, are released, which bind to capture antibodies. 3) Wash plate. Incubate with biotinylated detection antibody. 4) Wash plate. Incubate with streptavidin-alkaline phosphatase conjugated. 5)Incubate with BCIP/NBT. Precipitated product forms, results in blue/purple spots.

Protocol

StepProcedure
1.Add 100 µl of PBS 1X to every well.
2.Incubate plate at room temperature (RT) for 10 min.
2.Incubate plate at room temperature (RT) for 10 min.
3.Empty the wells by flicking the plate over a sink & gently tapping on absorbent paper.
3.Empty the wells by flicking the plate over a sink & gently tapping on absorbent paper.
4.Add 100 µl of sample, positive and negative controls cell suspension to appropriate wells providing the required concentration of cells and stimulant.
5.Cover the plate and incubate at 37°C in a CO2 incubator for an appropriate length of time (15-20 hours). Note: do not agitate or move the plate during this incubation.
6.Empty the wells and remove excess solution then add 100 µl of Wash Buffer to every well.
7.Incubate the plate at 4°C for 10 min.
8.Empty the wells as previous and wash the plate 3x with 100 µl of Wash Buffer.
9.Add 100 µl of diluted detection antibody to every well.
10.Cover the plate and incubate at RT for 1 hour 30 min.
11.Empty the wells as previous and wash the plate 3x with 100 µl of Wash Buffer.
12.Add 100 µl of diluted Streptavidin-AP conjugate to every well.
13.Cover the plate and incubate at RT for 1 hour.
14.Empty the wells and wash the plate 3x with 100 µl of Wash Buffer.
15.Peel of the plate bottom and wash both sides of the membrane 3x under running distilled water, once washing is complete remove any excess solution by repeated tapping on absorbent paper.
16.Add 100 µl of ready-to-use BCIP/NBT buffer to every well.
17.Incubate the plate for 5-15 min monitoring spot formation visually throughout the incubation period to assess sufficient colour development.
18.Empty the wells and rinse both sides of the membrane 3x under running distilled water. Completely remove any excess solution by gentle repeated tapping on absorbent paper.
19.Read Spots: allow the wells to dry and then read results. The frequency of the resulting coloured spots corresponding to the cytokine producing cells can be determined using an appropriate ELISpot reader and analysis software or manually using a microscope. Note: spots may become sharper after overnight incubation at 4°C in the dark.

Human CD178 ELISpot Pair - Information

Product type: ELISpot Pairs
Target species: Human
Specificity: Recognizes natural human CD178
Incubation:
Kit content: Assay Genie ELISpot matched antibody pairs are extensively validated and include pre-titrated capture antibody and biotinylated detection antibody. Antibodies are supplied in quantities sufficient for 10 x 96 samples.
Synonyms: FAS-Ligand, CD95L
Uniprot: P48023

CD178 - Protein Information

UniProt Protein Function:FasL: Cytokine that binds to TNFRSF6/FAS, a receptor that transduces the apoptotic signal into cells. May be involved in cytotoxic T-cell mediated apoptosis and in T-cell development. TNFRSF6/FAS-mediated apoptosis may have a role in the induction of peripheral tolerance, in the antigen-stimulated suicide of mature T-cells, or both. Binding to the decoy receptor TNFRSF6B/DcR3 modulates its effects. Homotrimer (Probable). Interacts with ARHGAP9, BAIAP2L1, BTK, CACNB3, CACNB4, CRK, DLG2, DNMBP, DOCK4, EPS8L3, FGR, FYB, FYN, HCK, ITK, ITSN2, KALRN, LYN, MACC1, MIA, MPP4, MYO15A, NCF1, NCK1, NCK2, NCKIPSD, OSTF1, PIK3R1, PSTPIP1, RIMBP3C, SAMSN1, SH3GL3, SH3PXD2B, SH3PXD2A, SH3RF2, SKAP2, SNX33, SNX9, SORBS3, SPTA1, SRC, SRGAP1, SRGAP2, SRGAP3, TEC, TJP3 and YES1. Belongs to the tumor necrosis factor family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Membrane protein, integral; Apoptosis; Cytokine

Chromosomal Location of Human Ortholog: 1q23

Cellular Component: extracellular space; lysosomal lumen; perinuclear region of cytoplasm; integral to plasma membrane; extracellular region; plasma membrane; caveola; nucleus; external side of plasma membrane

Molecular Function:protein binding; death receptor binding; cytokine activity; tumor necrosis factor receptor binding; receptor binding

Biological Process: caspase activation; positive regulation of I-kappaB kinase/NF-kappaB cascade; transcription, DNA-dependent; apoptosis; positive regulation of apoptosis; response to lipopolysaccharide; negative regulation of transcription from RNA polymerase II promoter; signal transduction; cellular chloride ion homeostasis; endosomal lumen acidification; inflammatory cell apoptosis; negative regulation of angiogenesis; induction of apoptosis via death domain receptors; cell-cell signaling; positive regulation of epidermal growth factor receptor signaling pathway; positive regulation of neuron apoptosis; positive regulation of cell proliferation; immune response; retinal cell programmed cell death

Disease: Lung Cancer; Autoimmune Lymphoproliferative Syndrome

NCBI Summary:This gene is a member of the tumor necrosis factor superfamily. The primary function of the encoded transmembrane protein is the induction of apoptosis triggered by binding to FAS. The FAS/FASLG signaling pathway is essential for immune system regulation, including activation-induced cell death (AICD) of T cells and cytotoxic T lymphocyte induced cell death. It has also been implicated in the progression of several cancers. Defects in this gene may be related to some cases of systemic lupus erythematosus (SLE). Alternatively spliced transcript variants have been described. [provided by RefSeq, Nov 2014]
UniProt Code:P48023
NCBI GenInfo Identifier:1345957
NCBI Gene ID:356
NCBI Accession:P48023.1
UniProt Secondary Accession:P48023,Q9BZP9,
UniProt Related Accession:P48023
Molecular Weight:14,006 Da
NCBI Full Name:Tumor necrosis factor ligand superfamily member 6
NCBI Synonym Full Names:Fas ligand (TNF superfamily, member 6)
NCBI Official Symbol:FASLG  
NCBI Official Synonym Symbols:APTL; FASL; CD178; CD95L; ALPS1B; CD95-L; TNFSF6; APT1LG1  
NCBI Protein Information:tumor necrosis factor ligand superfamily member 6; CD95 ligand; fas antigen ligand; apoptosis antigen ligand; apoptosis (APO-1) antigen ligand 1
UniProt Protein Name:Tumor necrosis factor ligand superfamily member 6
UniProt Synonym Protein Names:Apoptosis antigen ligand; APTL; CD95 ligand; CD95-L; Fas antigen ligand; Fas ligand; FasL; CD_antigen: CD178Cleaved into the following 4 chains:Tumor necrosis factor ligand superfamily member 6, membrane form; Tumor necrosis factor ligand superfamily member 6, soluble formAlternative name(s):Receptor-binding FasL ectodomain; Soluble Fas ligand; sFasL
Protein Family:S-fimbrial adhesin protein
UniProt Gene Name:FASLG  
UniProt Entry Name:TNFL6_HUMAN
View AllClose

Additional Information

Product type:
ELISpot
Reactivity:
Human
View AllClose