Product Name:	Human sTNF RI / TNFRSF1A ELISA Kit
Product Code:	HUFI00373
Size:	96 Assays
Alias:	sTNF RI, TNFRSF1A, TNF-RI, TNF-R-I, TNFR-I, CD120a, TNFRSF1A, CD120a, CD120a antigen, FPF, p55, p55-R, p60, TNFARMGC19588, TNF-R, TNF-R1, TNFR1TBP1, TNFR55, TNF-R55, TNFR60
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human TNFRSF1A concentrations in serum plasma and other biological fluids.
Sensitivity:	4.688pg/ml
Range:	7.813-500pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human TNFRSF1A and the recovery rates were calculated by comparing the measured value to the expected amount of Human TNFRSF1A in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	90-101	98
EDTA plasma(n=5)	91-100	95
UFH plasma(n=5)	89-104	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TNFRSF1A and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	92-105%	95-100%	85-97%
EDTA plasma(n=5)	88-99%	90-99%	87-96%
UFH plasma(n=5)	81-96%	81-96%	88-98%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P19438

UniProt Protein Function:	TNF-R1: Receptor for TNFSF2/TNF-alpha and homotrimeric TNFSF1/lymphotoxin-alpha. The adapter molecule FADD recruits caspase-8 to the activated receptor. The resulting death-inducing signaling complex (DISC) performs caspase-8 proteolytic activation which initiates the subsequent cascade of caspases (aspartate- specific cysteine proteases) mediating apoptosis. Contributes to the induction of non-cytocidal TNF effects including anti-viral state and activation of the acid sphingomyelinase. Binding of TNF to the extracellular domain leads to homotrimerization. The aggregated death domains provide a novel molecular interface that interacts specifically with the death domain of TRADD. Various TRADD-interacting proteins such as TRAFS, RIPK1 and possibly FADD, are recruited to the complex by their association with TRADD. This complex activates at least two distinct signaling cascades, apoptosis and NF-kappa-B signaling. Interacts with BAG4, BRE, FEM1B, GRB2, SQSTM1 and TRPC4AP. Interacts with HCV core protein. Interacts with human cytomegalovirus/HHV-5 protein UL138. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Receptor, cytokine; Membrane protein, integral Chromosomal Location of Human Ortholog: 12p13.2 Cellular Component: Golgi membrane; extracellular space; cell surface; mitochondrion; integral to plasma membrane; axon; extracellular region; plasma membrane; synapse; nucleus; cytosol; receptor complex; lipid raft Molecular Function:protein binding; tumor necrosis factor receptor activity; protease binding; protein complex binding; tumor necrosis factor binding Biological Process: response to alkaloid; viral reproduction; protein heterooligomerization; response to lipopolysaccharide; tumor necrosis factor-mediated signaling pathway; positive regulation of neuron apoptosis; negative regulation of interleukin-6 production; response to wounding; tetrapyrrole metabolic process; inflammatory response; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of protein import into nucleus, translocation; cytokine and chemokine mediated signaling pathway; positive regulation of tumor necrosis factor production; response to amino acid stimulus; prostaglandin metabolic process; positive regulation of tyrosine phosphorylation of Stat1 protein; positive regulation of angiogenesis; response to ethanol; induction of apoptosis via death domain receptors; DNA damage response, signal transduction resulting in induction of apoptosis; negative regulation of inflammatory response; defense response to bacterium; response to hypoxia; positive regulation of transcription from RNA polymerase II promoter; positive regulation of inflammatory response; negative regulation of apoptosis Disease: Multiple Sclerosis, Susceptibility To, 5; Periodic Fever, Familial, Autosomal Dominant
NCBI Summary:	The protein encoded by this gene is a member of the TNF-receptor superfamily. This protein is one of the major receptors for the tumor necrosis factor-alpha. This receptor can activate NF-kappaB, mediate apoptosis, and function as a regulator of inflammation. Antiapoptotic protein BCL2-associated athanogene 4 (BAG4/SODD) and adaptor proteins TRADD and TRAF2 have been shown to interact with this receptor, and thus play regulatory roles in the signal transduction mediated by the receptor. Germline mutations of the extracellular domains of this receptor were found to be associated with the autosomal dominant periodic fever syndrome. The impaired receptor clearance is thought to be a mechanism of the disease. [provided by RefSeq, Jul 2008]
UniProt Code:	P19438
NCBI GenInfo Identifier:	135959
NCBI Gene ID:	7132
NCBI Accession:	P19438.1
UniProt Secondary Accession:	P19438,Q9UCA4, A8K4X3, B2RDE4, B3KPQ1, B4DQB7, B4E309 B5M0B5, D3DUR1,
UniProt Related Accession:	P19438
Molecular Weight:	24,194 Da
NCBI Full Name:	Tumor necrosis factor receptor superfamily member 1A
NCBI Synonym Full Names:	tumor necrosis factor receptor superfamily, member 1A
NCBI Official Symbol:	TNFRSF1A
NCBI Official Synonym Symbols:	FPF; MS5; p55; p60; TBP1; TNF-R; TNFAR; TNFR1; p55-R; CD120a; TNFR55; TNFR60; TNF-R-I; TNF-R55; TNFR1-d2
NCBI Protein Information:	tumor necrosis factor receptor superfamily member 1A; TNF-R1; TNF-RI; TNFR-I; tumor necrosis factor-alpha receptor; tumor necrosis factor receptor type 1; tumor necrosis factor binding protein 1; tumor necrosis factor receptor 1A isoform beta
UniProt Protein Name:	Tumor necrosis factor receptor superfamily member 1A
UniProt Synonym Protein Names:	Tumor necrosis factor receptor 1; TNF-R1; Tumor necrosis factor receptor type I; TNF-RI; TNFR-I; p55; p60; CD_antigen: CD120aCleaved into the following 2 chains:Tumor necrosis factor receptor superfamily member 1A, membrane form; Tumor necrosis factor-binding protein 1; TBPI
UniProt Gene Name:	TNFRSF1A
UniProt Entry Name:	TNR1A_HUMAN

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 Âµl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 Âµl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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Human sTNF RI / TNFRSF1A ELISA Kit

Description