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Human AMFR / E3 ubiquitin-protein ligase ELISA Kit

SKU:
HUFI01400
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9UKV5
Sensitivity:
9.375pg/ml
Range:
15.625-1000pg/ml
ELISA Type:
Competitive
Synonyms:
AMFR, E3 ubiquitin-protein ligase AMFR, gp78, Autocrine motility factor receptor, AMF receptor, RING finger protein 45, GP78, RNF45
Reactivity:
Human
Research Area:
Signal Transduction
€599
Frequently bought together:

Description

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Human AMFR / E3 ubiquitin-protein ligase ELISA

AMFR (Autocrine Motility Factor Receptor) locus encodes a glycosylated transmembrane receptor AMFR inhibitors are a potential new treatment for cancer. They work by blocking the action of a protein called c-Met, which helps cancer cells to grow and spread. Diseases associated with AMFR include Hepatocellular Clear Cell Carcinoma and High-Grade Astrocytoma. Among its related pathways are Metabolism of proteins and Regulation of degradation of deltaF508 CFTR in CF. An important paralog of AMFR is SYVN1.

Product Name:Human AMFR / E3 ubiquitin-protein ligase ELISA Kit
Product Code:HUFI01400
Size:96 Assays
Alias:AMFR, E3 ubiquitin-protein ligase AMFR, gp78, Autocrine motility factor receptor, AMF receptor, RING finger protein 45, GP78, RNF45
Detection method:Competitive ELISA, Coated with Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human AMFR concentrations in serum plasma and other biological fluids.
Sensitivity:9.375pg/ml
Range:15.625-1000pg/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human AMFR and the recovery rates were calculated by comparing the measured value to the expected amount of Human AMFR in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)86-10598
EDTA plasma(n=5)86-10192
UFH plasma(n=5)85-10394
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human AMFR and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)88-100%85-98%90-101%
EDTA plasma(n=5)91-97%84-101%90-101%
UFH plasma(n=5)80-98%80-87%85-96%
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)60ul4°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 pipettetips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniprotQ9UKV5
UniProt Protein Function:AMFR: E3 ubiquitin-protein ligase that mediates the polyubiquitination of a number of proteins such as CD3D, CYP3A4, CFTR and APOB for proteasomal degradation. Component of a VCP/p97- AMFR/gp78 complex that participates in the final step of endoplasmic reticulum-associated degradation (ERAD). The VCP/p97- AMFR/gp78 complex is involved in the sterol-accelerated ERAD degradation of HMGCR through binding to the HMGCR-INSIG complex at the ER membrane and initiating ubiquitination of HMGCR. The ubiquitinated HMGCR is then released from the ER by the complex into the cytosol for subsequent destruction. Also acts as a scaffold protein to assemble a complex that couples ubiquitination, retranslocation and deglycosylation. Mediates tumor invasion and metastasis. Interacts with RNF5. Also forms an ERAD complex containing VCP/p97, NGLY1; PSMC1; SAKS1 AND RAD23B required for coupling retrotranslocation, ubiquitination and deglycosylation. Interacts with DRL1. Interacts (through a region distinct from the RING finger) with UBE2G2/UBC7. Component of the VCP/p97-AMFR/gp78 complex that enhances VCP/p97 binding to polyubiquitinated proteins for their degradation by the endoplasmic reticulum-associated degradation (ERAD) pathway. Interacts (via the VIM) with VCP/p97. Interacts (via its membrane domain) with INSIG1; the interaction initiates the sterol-mediated ubiquitination and degradation of HMGCR by the ERAD pathway. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:Protein type:Ubiquitin ligase; Ligase; Membrane protein, integral; EC 6.3.2.19; Endoplasmic reticulum; Ubiquitin conjugating system; Motility/polarity/chemotaxis; EC 6.3.2.-; Membrane protein, multi-pass; Receptor, misc.

Chromosomal Location of Human Ortholog: 16q21

Cellular Component: endoplasmic reticulum membrane; growth cone; protein complex; cell soma; membrane; perinuclear region of cytoplasm; dendrite; integral to membrane; integral to endoplasmic reticulum membrane; nucleus

Molecular Function:protein binding; zinc ion binding; ubiquitin-protein ligase activity; receptor activity; ligase activity

Biological Process: ubiquitin-dependent protein catabolic process; ER-associated protein catabolic process; protein polyubiquitination; learning and/or memory; unfolded protein response; cell motility; signal transduction; protein oligomerization; aging

NCBI Summary:This locus encodes a glycosylated transmembrane receptor. Its ligand, autocrine motility factor, is a tumor motility-stimulating protein secreted by tumor cells. The encoded receptor is also a member of the E3 ubiquitin ligase family of proteins. It catalyzes ubiquitination and endoplasmic reticulum-associated degradation of specific proteins. [provided by RefSeq, Feb 2012]
UniProt Code:Q9UKV5
NCBI GenInfo Identifier:113707
NCBI Gene ID:267
NCBI Accession:P26442.1
UniProt Secondary Accession:Q9UKV5,P26442, Q8IZ70,
UniProt Related Accession:Q9UKV5
Molecular Weight:323
NCBI Full Name:
NCBI Synonym Full Names:autocrine motility factor receptor
NCBI Official Symbol:AMFR
NCBI Official Synonym Symbols:GP78; RNF45
NCBI Protein Information:E3 ubiquitin-protein ligase AMFR
UniProt Protein Name:E3 ubiquitin-protein ligase AMFR
UniProt Synonym Protein Names:Autocrine motility factor receptor; AMF receptor; RING finger protein 45; gp78
Protein Family:E3 ubiquitin-protein ligase
UniProt Gene Name:AMFR
UniProt Entry Name:AMFR_HUMAN

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

Equilibrate the TMB substrate for at least 30 min at 37°C beforeuse. When diluting samples and reagents, they must be mixed completely andevenly. It is recommended to plot a standard curve for each test.

StepProtocol
1.Set standard, test sample and control (zero) wells on the pre-coatedplate respectively, and then, record their positions. It isrecommended to measure each standard and sample in duplicate. Washplate 2 times before adding standard, sample and control (zero) wells!
2.Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blankwell is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody workingsolution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thoroughmixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).
3.Wash: Aspirate each well and wash, repeating the process three timesWash by filling each well with Wash Buffer (approximately 350µL)using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essentialto good performance. After the last wash, remove any remaining WashBuffer by aspirating or decanting. Invert the plate and pat it againstthick clean absorbent paper.
4.HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC workingsolution to each well. Cover with a new Plate sealer. Incubate for30minutes at 37°C.
5.Wash: Repeat the aspiration/wash process for five times.
6.TMB Substrate: Add 90µL of TMB Substrate to each well. Coverwith a new Plate sealer. Incubate for about 10-20 minutes at 37°C.Protect from light. The reaction time can be shortened or extendedaccording to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, you can terminatethe reaction.
7.Stop: Add 50µL of Stop Solution to each well. Color turn toyellow immediately. The adding order of stop solution should be as thesame as the substrate solution.
8.OD Measurement: Determine the optical density (OD Value) of each wellat once, using a microplate reader set to 450 nm. You should open themicroplate reader ahead, preheat the instrument, and set the testing parameters.

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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