Human TNFRSF1A ELISA Kit (HUEB0155)- High Sensitivity

Product Name:	Human Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A) ELISA Kit
SKU:	HUEB0155
Size:	96T
Target:	Human Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A)
Synonyms:	Tumor necrosis factor receptor 1, Tumor necrosis factor receptor type I, p55, p60, TNF-R1, TNF-RI, CD120a, TNFAR, TNFR1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	15.6-1000pg/mL
Sensitivity:	10pg/mL

Intra CV:

4.9%

Inter CV:

8.0%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	104-114%	104-114%	100-113%	106-118%
EDTA Plasma(N=5)	95-104%	94-106%	100-111%	115-124%
Heparin Plasma(N=5)	100-109%	111-120%	105-116%	98-108%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	89	83-95
Plasma	91	85-97

Function:

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.

Uniprot:	P19438
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Tumor necrosis factor receptor superfamily member 1A
Sub Unit:	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. Interacts directly with NOL3 (via CARD domain); inhibits TNF-signaling pathway.
Research Area:	Neurosciences
Subcellular Location:	Isoform 4 Secreted Lacks a Golgi-retention motif, is not membrane bound and therefore is secreted.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

ELISA	Antibodies
Human sTNF RI / TNFRSF1A ELISA Kit	Anti-TNFRSF1A Antibody (CAB0033)
Human TNFRSF1A (Tumor Necrosis Factor Receptor Superfamily, Member 1A) CLIA Kit (HUES00176)	Anti-TNFR1 Antibody (CAB1540)
Human TNFRSF1A (Tumor Necrosis Factor Receptor Superfamily, Member 1A) ELISA Kit (HUES01474)	TNFAIP6 Antibody (PACO05219)
	PYCARD Antibody (PACO13641)
	HAT1 Antibody (PACO21666)