Human sTNF RI / TNFRSF1A ELISA Kit
Human sTNF RI / TNFRSF1A ELISA Kit - Information
The ELISA Genie sTNF RI / TNFRSF1A ELISA Kit can assay for sTNF RI / TNFRSF1A in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.How our sTNF RI / TNFRSF1A ELISA Kits Work?
The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.
At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound sTNF RI / TNFRSF1A is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.
Human sTNF RI / TNFRSF1A ELISA Kit - Data
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.
|Post-Translational Modification|| |
The soluble form is produced from the membrane form by proteolytic processing.
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
This immunoassay kit allows for the in vitro quantitative determination of TNFRSF1A concentrations in serum plasma and other biological fluids.
4'C for 6 months
Matrices listed below were spiked with certain level of TNFRSF1A and the recovery rates were calculated by comparing the measured value to the expected amount of TNFRSF1A in samples.
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of TNFRSF1A and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
For Research Use Only
Human sTNF RI / TNFRSF1A ELISA Kit Protocol
The below protocol is a sample protocol for Human sTNF RI / TNFRSF1A ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human sTNF RI / TNFRSF1A present in their sample.
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.
|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 µl 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 µl 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.|
Human sTNF RI / TNFRSF1A ELISA Kit components
|ELISA Microplate(Dismountable)||8×12 strips||4°C for 6 months|
|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)|
Other materials and equipment required:The ELISA Genie Human sTNF RI / TNFRSF1A ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
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.
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.
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.
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.
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.
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.
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.
Human sTNF RI / TNFRSF1A ELISA Kit Protein Information
|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]|
|NCBI GenInfo Identifier:||135959|
|NCBI Gene ID:||7132|
|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|