Human Angiopoietin-like 3 / ANGPTL3 ELISA Kit
Human Angiopoietin-like 3 / ANGPTL3 ELISA Kit - Information
The ELISA Genie Angiopoietin-like 3 / ANGPTL3 ELISA Kit can assay for Angiopoietin-like 3 / ANGPTL3 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.How our Angiopoietin-like 3 / ANGPTL3 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 Angiopoietin-like 3 / ANGPTL3 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 Angiopoietin-like 3 / ANGPTL3 ELISA Kit - Data
Acts in part as a hepatokine that is involved in regulation of lipid and glucose metabolism (PubMed:11788823, PubMed:12909640, PubMed:23661675, PubMed:25495645). Proposed to play a role in the trafficking of energy substrates to either storage or oxidative tissues in response to food intake. Has a stimulatory effect on plasma triglycerides (TG), which is achieved by suppressing plasma TG clearance via inhibition of LPL activity. The inhibition of LPL activity appears to be an indirect mechanism involving recruitment of proprotein convertases PCSK6 and FURIN to LPL leading to cleavage and dissociation of LPL from the cell surface; the function does not require ANGPTL3 proteolytic cleavage but seems to be mediated by the N-terminal domain, and is not inhibited by GPIHBP1 (PubMed:12097324, PubMed:19318355, PubMed:20581395). Can inhibit endothelial lipase, causing increased plasma levels of high density lipoprotein (HDL) cholesterol and phospholipids (PubMed:17110602, PubMed:19028676). Can bind to adipocytes to activate lipolysis, releasing free fatty acids and glycerol (PubMed:12565906). Suppresses LPL specifically in oxidative tissues which is required to route very low density lipoprotein (VLDL)-TG to white adipose tissue (WAT) for storage in response to food; the function may involve cooperation with circulating, liver-derived ANGPTL8 and ANGPTL4 expression in WAT. Contributes to lower plasma levels of low density lipoprotein (LDL)-cholesterol by a mechanism that is independent of the canonical pathway implicating APOE and LDLR. May stimulate hypothalamic LPL activity.; ANGPTL3(17-221): In vitro inhibits LPL activity; not effective on GPIHBP1-stabilized LPL.; Involved in angiogenesis. Binds to endothelial cells via integrin alpha-V/beta-3 (ITGAV:ITGB3), activates FAK, MAPK and Akt signaling pathways and induces cell adhesion and cell migration (PubMed:11877390). Secreted from podocytes, may modulate properties of glomerular endothelial cells involving integrin alpha-V/beta-3 and Akt signaling (PubMed:18535744). May increase the motility of podocytes. May induce actin filament rearrangements in podocytes implicating integrin alpha-V/beta-3 and Rac1 activation. Binds to hematopoietic stem cells (HSC) and is involved in the regulation of HSC activity probably implicating down-regulation of IKZF1/IKAROS.
|Post-Translational Modification|| |
O-glycosylated at Thr-226 by GALNT2; blocks processing and activation by proprotein convertases. In part proteolytically cleaved by proprotein convertases; proposed to be involved in activation.
ANGPTL3(Angiopoietin Like Protein 3)/AGNPT5/Ang-5/Angiopoietin-5/ANG-5/angiopoietin 5/angiopoietin-like 3/Angiopoietin-like protein 3/angiopoietin-related protein 3/ANGPT5angiopoietin-5/FHBL2
|Detection method|| |
Sandwich ELISA Double Antibody
This immunoassay kit allows for the in vitro quantitative determination of ANGPTL3 concentrations in serum plasma and other biological fluids.
4'C for 6 months
Matrices listed below were spiked with certain level of ANGPTL3 and the recovery rates were calculated by comparing the measured value to the expected amount of ANGPTL3 in samples.
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of ANGPTL3 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
For Research Use Only
Human Angiopoietin-like 3 / ANGPTL3 ELISA Kit Protocol
The below protocol is a sample protocol for Human Angiopoietin-like 3 / ANGPTL3 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 Angiopoietin-like 3 / ANGPTL3 present in their sample.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 Â°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 Â°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 37Â°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 37Â°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 37Â°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.|
Human Angiopoietin-like 3 / ANGPTL3 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 Angiopoietin-like 3 / ANGPTL3 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 Angiopoietin-like 3 / ANGPTL3 ELISA Kit Protein Information
|UniProt Protein Function:||ANGPTL3: Defects in ANGPTL3 are the cause of familial hypobetalipoproteinemia type 2 (FHBL2); also called combined hypobetalipoproteinemia familial. FHBL2 is a disorder of lipid metabolism characterized by less than 5th percentile age- and sex-specific levels of low density lipoproteins, and dietary fat malabsorption. Affected individuals present with combined hypolipidemia, consisting of extremely low plasma levels of LDL cholesterol, HDL cholesterol, and triglycerides.|
|UniProt Protein Details:|
Protein type:Secreted; Cell adhesion; Inhibitor; Motility/polarity/chemotaxis; Secreted, signal peptide
Chromosomal Location of Human Ortholog: 1p31.3
Cellular Component: extracellular space; cell surface
Molecular Function:integrin binding; enzyme inhibitor activity; phospholipase inhibitor activity; growth factor activity
Biological Process: integrin-mediated signaling pathway; cholesterol metabolic process; phospholipid catabolic process; glycerol metabolic process; cell-matrix adhesion; lipid homeostasis; positive regulation of lipid catabolic process; fatty acid metabolic process; signal transduction; sequestering of lipid; positive regulation of angiogenesis; cholesterol homeostasis; acylglycerol homeostasis; phospholipid metabolic process; artery morphogenesis; negative regulation of lipoprotein lipase activity; positive regulation of cell migration; phospholipid homeostasis
Disease: Hypobetalipoproteinemia, Familial, 2
|NCBI Summary:||This gene encodes a member of a family of secreted proteins that function in angiogenesis. The encoded protein, which is expressed predominantly in the liver, is further processed into an N-terminal coiled-coil domain-containing chain and a C-terminal fibrinogen chain. The N-terminal chain is important for lipid metabolism, while the C-terminal chain may be involved in angiogenesis. Mutations in this gene cause familial hypobetalipoproteinemia type 2. [provided by RefSeq, Aug 2015]|
|NCBI GenInfo Identifier:||25008126|
|NCBI Gene ID:||27329|
|UniProt Related Accession:||Q9Y5C1|
|Molecular Weight:||~ 54kDa|
|NCBI Full Name:||Angiopoietin-related protein 3|
|NCBI Synonym Full Names:||angiopoietin like 3|
|NCBI Official Symbol:||ANGPTL3|
|NCBI Official Synonym Symbols:||ANL3; ANG-5; FHBL2; ANGPT5|
|NCBI Protein Information:||angiopoietin-related protein 3|
|UniProt Protein Name:||Angiopoietin-related protein 3|
|UniProt Synonym Protein Names:||Angiopoietin-5; ANG-5; Angiopoietin-like protein 3|
|Protein Family:||Angiopoietin-related protein|
|UniProt Gene Name:||ANGPTL3|
|UniProt Entry Name:||ANGL3_HUMAN|