Description
| Product Name: | Human Apolipoprotein A-I (APOA1) ELISA Kit |
| Product Code: | HUEB0201 |
| Alias: | Apolipoprotein A-I, Apo-AI, ApoA-I, Apolipoprotein A1, APOA1 |
| Uniprot: | P02647 |
| Reactivity: | Human |
| Range: | 7.8-500 ng/mL |
| Detection Method: | Sandwich |
| Size: | 96 Assay |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | APOA1: Participates in the reverse transport of cholesterol from tissues to the liver for excretion by promoting cholesterol efflux from tissues and by acting as a cofactor for the lecithin cholesterol acyltransferase (LCAT). As part of the SPAP complex, activates spermatozoa motility. Interacts with APOA1BP and CLU. Component of a sperm activating protein complex (SPAP), consisting of APOA1, an immunoglobulin heavy chain, an immunoglobulin light chain and albumin. Interacts with NDRG1. Major protein of plasma HDL, also found in chylomicrons. Synthesized in the liver and small intestine. The oxidized form at Met-110 and Met-136 is increased in individuals with increased risk for coronary artery disease, such as in carrier of the eNOSa/b genotype and exposure to cigarette smoking. It is also present in increased levels in aortic lesions relative to native ApoA-I and increased levels are seen with increasing severity of disease. Belongs to the apolipoprotein A1/A4/E family. |
| UniProt Protein Details: | Protein type:Lipid-binding; Secreted, signal peptide; Secreted; Vesicle; Motility/polarity/chemotaxis; Cell development/differentiation; Endoplasmic reticulum Chromosomal Location of Human Ortholog: 11q23-q24 Cellular Component: extracellular space; chylomicron; cell surface; endocytic vesicle; endoplasmic reticulum lumen; early endosome; extracellular region; plasma membrane; cytoplasmic vesicle; nucleus; cytosol; vesicle Molecular Function:identical protein binding; protein binding; enzyme binding; phospholipid transporter activity; lipase inhibitor activity; beta-amyloid binding; chemorepellent activity; cholesterol transporter activity; cholesterol binding; phospholipid binding; phosphatidylcholine binding; apolipoprotein A-I receptor binding; high-density lipoprotein binding; apolipoprotein receptor binding Biological Process: phototransduction, visible light; negative chemotaxis; negative regulation of lipase activity; axon regeneration in the peripheral nervous system; sequestering of lipid; negative regulation of interleukin-1 beta secretion; regulation of cholesterol absorption; transforming growth factor beta receptor signaling pathway; positive regulation of stress fiber formation; response to drug; platelet activation; cholesterol metabolic process; organ regeneration; regulation of Cdc42 protein signal transduction; adrenal gland development; positive regulation of hydrolase activity; positive regulation of Rho protein signal transduction; lipoprotein metabolic process; positive regulation of transferase activity; vitamin transport; cholesterol biosynthetic process; negative regulation of cytokine secretion during immune response; cholesterol homeostasis; lipoprotein biosynthetic process; response to estrogen stimulus; peptidyl-methionine modification; phosphatidylcholine biosynthetic process; positive regulation of lipoprotein lipase activity; blood vessel endothelial cell migration; cellular lipid metabolic process; platelet degranulation; phospholipid efflux; retinoid metabolic process; transmembrane transport; response to nutrient; phospholipid homeostasis; integrin-mediated signaling pathway; receptor-mediated endocytosis; positive regulation of fatty acid biosynthetic process; regulation of protein amino acid phosphorylation; cholesterol transport; protein stabilization; protein amino acid oxidation; negative regulation of heterotypic cell-cell adhesion; neurite regeneration; cholesterol efflux; G-protein coupled receptor protein signaling pathway; glucocorticoid metabolic process; reverse cholesterol transport; endothelial cell proliferation; negative regulation of inflammatory response; blood coagulation Disease: Hypoalphalipoproteinemia, Primary; Amyloidosis, Familial Visceral |
| NCBI Summary: | This gene encodes apolipoprotein A-I, which is the major protein component of high density lipoprotein (HDL) in plasma. The protein promotes cholesterol efflux from tissues to the liver for excretion, and it is a cofactor for lecithin cholesterolacyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters. This gene is closely linked with two other apolipoprotein genes on chromosome 11. Defects in this gene are associated with HDL deficiencies, including Tangier disease, and with systemic non-neuropathic amyloidosis. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P02647 |
| NCBI GenInfo Identifier: | 113992 |
| NCBI Gene ID: | 335 |
| NCBI Accession: | P02647.1 |
| UniProt Secondary Accession: | P02647,Q6LDN9, Q6Q785, Q9UCS8, Q9UCT8, A8K866, |
| UniProt Related Accession: | P02647 |
| Molecular Weight: | 30,778 Da |
| NCBI Full Name: | Apolipoprotein A-I |
| NCBI Synonym Full Names: | apolipoprotein A-I |
| NCBI Official Symbol: | APOA1 |
| NCBI Protein Information: | apolipoprotein A-I; apo-AI |
| UniProt Protein Name: | Apolipoprotein A-I |
| UniProt Synonym Protein Names: | Apolipoprotein A1Cleaved into the following 2 chains:Proapolipoprotein A-I; ProapoA-I; Truncated apolipoprotein A-IAlternative name(s):Apolipoprotein A-I(1-242) |
| Protein Family: | Apolipoprotein |
| UniProt Gene Name: | APOA1 |
| UniProt Entry Name: | APOA1_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. |
