Human FASL/TNFSF6 (Factor Related Apoptosis Ligand) ELISA Kit (HUES01355)

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HUES01355
€539

Description

ELISA Kit Technical ManualMSDS

Human FASL/TNFSF6 (Factor Related Apoptosis Ligand) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human FASL/TNFSF6 . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human FASL/TNFSF6 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human FASL/TNFSF6, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human FASL/TNFSF6. The concentration of Human FASL/TNFSF6 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range15.63-1000 pg/mL
Sensitivity9.38 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human FASL/TNFSF6 in samples. No significant cross-reactivity or interference between Human FASL/TNFSF6 and analogues was observed.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(pg/mL)
O.D Average Corrected
10002.457
2.461
2.4592.4
5001.629
1.667
1.6481.589
2500.905
0.891
0.8980.839
1250.442
0.448
0.4450.386
62.50.242
0.238
0.240.181
31.250.174
0.15
0.1620.103
15.630.107
0.117
0.1120.053
00.051
0.067
0.059--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human FASL/TNFSF6 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human FASL/TNFSF6 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)50.15142.30454.7550.49135.64412.66
Standard deviation2.948.2414.922.906.4715.76
C V (%)5.865.793.285.744.773.82

Recovery

The recovery of Human FASL/TNFSF6 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)95-108102
EDTA plasma (n=5)93-109100
Cell culture media (n=5)87-9994

Linearity

Samples were spiked with high concentrations of Human FASL/TNFSF6 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)97-11193-10690-103
Average (%)1039996
1:4Range (%)90-10681-9185-97
Average (%)988692
1:8Range (%)88-9988-10081-93
Average (%)949387
1:16Range (%)89-10382-9582-96
Average (%)968988

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human FASL/TNFSF6 (Factor Related Apoptosis Ligand) ELISA Kit (HUES01355) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human FASL/TNFSF6 (Factor Related Apoptosis Ligand) ELISA Kit (HUES01355) 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
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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