Human TRAF6 / TNF receptor-associated factor 6 ELISA Kit

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SKU:
HUFI02052
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9Y4K3
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
TRAF6, RNF85, MGC:3310, RING finger protein 85, E3 ubiquitin-protein ligase TRAF6, Interleukin-1 signal transducer, E3 ubiquitin-protein ligase TRAF6, EC 6.3.2.-, Interleukin-1 signal transducer, RING finger protein 85, RNF85MGC:3310, TNF receptor-as
Reactivity:
Human
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Description

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Product Name:Human TRAF6 / TNF receptor-associated factor 6 ELISA Kit
Product Code:HUFI02052
Size:96 Assays
Alias:TRAF6, RNF85, MGC:3310, RING finger protein 85, E3 ubiquitin-protein ligase TRAF6, Interleukin-1 signal transducer, E3 ubiquitin-protein ligase TRAF6, EC 6.3.2.-, Interleukin-1 signal transducer, RING finger protein 85, RNF85MGC:3310, TNF receptor-associated factor 6
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human TRAF6 concentrations in serum plasma and other biological fluids.
Sensitivity:0.094ng/ml
Range:0.156-10ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human TRAF6 and the recovery rates were calculated by comparing the measured value to the expected amount of Human TRAF6 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)85-10496
EDTA plasma(n=5)86-10496
UFH plasma(n=5)85-10493
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TRAF6 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)87-101%87-98%92-101%
EDTA plasma(n=5)84-101%84-98%89-101%
UFH plasma(n=5)80-100%81-91%85-99%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

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
UniprotQ9Y4K3
UniProt Protein Function:TRAF6: E3 ubiquitin ligase that, together with UBE2N and UBE2V1, mediates the synthesis of 'Lys-63'-linked-polyubiquitin chains conjugated to proteins, such as IKBKG, AKT1 and AKT2. Also mediates ubiquitination of free/unanchored polyubiquitin chain that leads to MAP3K7 activation. Leads to the activation of NF- kappa-B and JUN. May be essential for the formation of functional osteoclasts. Seems to also play a role in dendritic cells (DCs) maturation and/or activation. Represses c-Myb-mediated transactivation, in B-lymphocytes. Adapter protein that seems to play a role in signal transduction initiated via TNF receptor, IL- 1 receptor and IL-17 receptor. Regulates osteoclast differentiation by mediating the activation of adapter protein complex 1 (AP-1) and NF-kappa-B, in response to RANK-L stimulation. Homotrimer. Homooligomer. N-terminal region is dimeric while C-terminal region is trimeric; maybe providing a mode of oligomerization. Binds to TNFRSF5/CD40 and TNFRSF11A/RANK. Associates with NGFR, TNFRSF17, IRAK1, IRAK2, IRAK3, IRAK4, RIPK2, MAP3K1, MAP3K5, MAP3K14, CSK, TRAF, TRAF-interacting protein TRIP and TNF receptor associated protein TDP2. Interacts with IL17R. Interacts with SQSTM1 bridging NTRK1 and NGFR. Forms a ternary complex with SQSTM1 and PRKCZ. Interacts with PELI1, PELI2 and PELI3. Binds UBE2V1. Interacts with MAVS/IPS1. Interacts with TAX1BP1. Interacts with IL1RL1. Interacts with TRAFD1. Interacts with ZNF675. Interacts with AJUBA. Interacts with TICAM1 and TICAM2. Interacts with ZFAND5. Interacts with ARRB1 and ARRB2. Interacts with MAP3K7 and TAB1/MAP3K7IP1; during IL-1 signaling. Interacts with UBE2N. Interacts with TGFBR1, HDAC1 and RANGAP1. Interacts with AKT1, AKT2 and AKT3. Interacts (via TRAF domains) with NUMBL (via C-terminal). Interacts (via TRAF domains) with WDR34 (via WD domains). Interacts with RBCK1. Interacts with TRAF3IP2. Interacts with LIMD1 (via LIM domains). Expressed in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Belongs to the TNF receptor-associated factor family. A subfamily.
UniProt Protein Details:Protein type:EC 6.3.2.-; Ligase; Ubiquitin conjugating system; Ubiquitin ligase

Chromosomal Location of Human Ortholog: 11p12

Cellular Component: cytoplasm; cytosol; endosome membrane; internal side of plasma membrane; lipid particle; mitochondrion; nucleolus; nucleus; perinuclear region of cytoplasm; plasma membrane; protein complex

Molecular Function:histone deacetylase binding; identical protein binding; mitogen-activated protein kinase kinase kinase binding; protein binding; protein kinase B binding; protein kinase binding; protein N-terminus binding; thioesterase binding; tumor necrosis factor receptor binding; ubiquitin conjugating enzyme binding; ubiquitin protein ligase binding; ubiquitin-protein ligase activity

Biological Process: activation of MAPK activity; activation of NF-kappaB transcription factor; activation of NF-kappaB-inducing kinase; activation of protein kinase activity; I-kappaB kinase/NF-kappaB cascade; JNK cascade; membrane protein intracellular domain proteolysis; MyD88-dependent toll-like receptor signaling pathway; MyD88-independent toll-like receptor signaling pathway; negative regulation of apoptosis; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; positive regulation of apoptosis; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of interleukin-2 production; positive regulation of JNK activity; positive regulation of osteoclast differentiation; positive regulation of T cell activation; positive regulation of T cell cytokine production; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; protein autoubiquitination; protein polyubiquitination; stimulatory C-type lectin receptor signaling pathway; T cell receptor signaling pathway; toll-like receptor 9 signaling pathway; toll-like receptor signaling pathway

NCBI Summary:The protein encoded by this gene is a member of the TNF receptor associated factor (TRAF) protein family. TRAF proteins are associated with, and mediate signal transduction from, members of the TNF receptor superfamily. This protein mediates signaling from members of the TNF receptor superfamily as well as the Toll/IL-1 family. Signals from receptors such as CD40, TNFSF11/RANCE and IL-1 have been shown to be mediated by this protein. This protein also interacts with various protein kinases including IRAK1/IRAK, SRC and PKCzeta, which provides a link between distinct signaling pathways. This protein functions as a signal transducer in the NF-kappaB pathway that activates IkappaB kinase (IKK) in response to proinflammatory cytokines. The interaction of this protein with UBE2N/UBC13, and UBE2V1/UEV1A, which are ubiquitin conjugating enzymes catalyzing the formation of polyubiquitin chains, has been found to be required for IKK activation by this protein. This protein also interacts with the transforming growth factor (TGF) beta receptor complex and is required for Smad-independent activation of the JNK and p38 kinases. This protein has an amino terminal RING domain which is followed by four zinc-finger motifs, a central coiled-coil region and a highly conserved carboxyl terminal domain, known as the TRAF-C domain. Two alternatively spliced transcript variants, encoding an identical protein, have been reported. [provided by RefSeq, Feb 2012]
UniProt Code:Q9Y4K3
NCBI GenInfo Identifier:30580642
NCBI Gene ID:7189
NCBI Accession:Q9Y4K3.1
UniProt Secondary Accession:Q9Y4K3,Q8NEH5, A6NKI7, A8KAB3, D3DR16,
UniProt Related Accession:Q9Y4K3
Molecular Weight:59,573 Da
NCBI Full Name:TNF receptor-associated factor 6
NCBI Synonym Full Names:TNF receptor associated factor 6
NCBI Official Symbol:TRAF6
NCBI Official Synonym Symbols:RNF85; MGC:3310
NCBI Protein Information:TNF receptor-associated factor 6
UniProt Protein Name:TNF receptor-associated factor 6
UniProt Synonym Protein Names:E3 ubiquitin-protein ligase TRAF6; Interleukin-1 signal transducer; RING finger protein 85; RING-type E3 ubiquitin transferase TRAF6Curated
Protein Family:TNF receptor-associated factor
UniProt Gene Name:TRAF6

*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.

StepProtocol
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.

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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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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