Human Agrin (AGRN) ELISA Kit (HUEB1368)
- SKU:
- HUEB1368
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- O00468
- Range:
- 0.156-10 ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- AGRN, Agrin, agrin proteoglycan
- Reactivity:
- Human
Description
Human Agrin (AGRN) ELISA Kit
The Human Agrin (AGRN) ELISA Kit is specially designed for the precise measurement of agrin levels in human serum, plasma, and cell culture supernatants. With its exceptional sensitivity and specificity, this kit delivers accurate and consistent results, making it perfect for various research purposes. Agrin is a key protein that plays a vital role in regulating synapse formation and maintenance, making it essential for proper neuromuscular junction function. Dysregulation of agrin has been linked to various neurological disorders, including myasthenia gravis and muscular dystrophy, highlighting its importance as a potential biomarker for studying and treating these conditions.
Elevate your research with the Human Agrin ELISA Kit and uncover valuable insights into the role of agrin in neurobiology and neurological diseases. This kit is a valuable tool for researchers looking to advance their understanding of agrin biology and its implications for human health.
| Product Name: | Human Agrin (AGRN) ELISA Kit |
| SKU: | HUEB1368 |
| Size: | 96T |
| Target: | Human Agrin (AGRN) |
| Synonyms: | AGRIN |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.156-10ng/mL |
| Sensitivity: | 0.078ng/mL |
| Intra CV: | 6.2% | ||||||||||||||||||||
| Inter CV: | 9.6% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Agrin C-terminal 22 kDa fragment: this released fragment is important for agrin signaling and to exert a maximal dendritic filopodia-inducing effect. All 'z' splice variants (z+) of this fragment also show an increase in the number of filopodia. |
| Uniprot: | O00468 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Agrin |
| Sub Unit: | Monomer (By similarity). Interacts (N-terminal subunit) with TGF-beta family members, BMP2 AND BMP4; the interactions inhibit the activity of these growth factors. Interacts with TGFB1; the interaction enhances the activity of TGFB1 (By similarity). Component of the AGRN-LRP4 complex that consists of a tetramer of two AGRN-LRP4 heterodimers. Interacts (via the laminin G-like 3 domain) directly with LRP4; the interaction is required for activation of MUSK and clustering of AChR and requires the 'z8' insert present in the z(+8) isoforms. Interacts with DAG1; the interaction is influenced by cell surface glycosaminoglycans and by alternative splicing of AGRN. |
| Research Area: | Neurosciences |
| Subcellular Location: | Isoform 2 Cell junction Synapse Cell membrane Single-pass type II membrane protein |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | agrin: Plays a central role in the formation and the maintenance of the neuromuscular junction (NMJ), the synapse between motor neuron and skeletal muscle. Ligand of the MUSK signaling complex that directly binds LRP4 in this complex and induces the phosphorylation of MUSK, the kinase of the complex. The activation of MUSK in myotubes induces the formation of NMJ by regulating different processes including the transcription of specific genes and the clustering of AChR in the postsynaptic membrane. Defects in AGRN are a cause of myasthenia, limb-girdle, familial (LGM). A congenital myasthenic syndrome characterized by a typical 'limb girdle' pattern of muscle weakness with small, simplified neuromuscular junctions but normal acetylcholine receptor and acetylcholinesterase function. |
| UniProt Protein Details: | Protein type:Cell adhesion; Cell development/differentiation; Extracellular matrix; Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 1p36.33 Cellular Component: basal lamina; cell junction; cytosol; extracellular matrix; extracellular region; Golgi lumen; integral component of membrane; lysosomal lumen; plasma membrane; synapse Molecular Function:calcium ion binding; heparan sulfate proteoglycan binding; laminin binding; protein binding; sialic acid binding; structural constituent of cytoskeleton Biological Process: clustering of voltage-gated sodium channels; cytoskeleton organization; extracellular matrix organization; G-protein coupled acetylcholine receptor signaling pathway; glycosaminoglycan biosynthetic process; glycosaminoglycan catabolic process; glycosaminoglycan metabolic process; positive regulation of filopodium formation; positive regulation of GTPase activity; positive regulation of synaptic growth at neuromuscular junction; positive regulation of transcription from RNA polymerase II promoter; receptor clustering; retinoid metabolic process; signal transduction; synapse organization Disease: Myasthenic Syndrome, Congenital, With Pre- And Postsynaptic Defects |
| NCBI Summary: | This gene encodes one of several proteins that are critical in the development of the neuromuscular junction (NMJ), as identified in mouse knock-out studies. The encoded protein contains several laminin G, Kazal type serine protease inhibitor, and epidermal growth factor domains. Additional post-translational modifications occur to add glycosaminoglycans and disulfide bonds. In one family with congenital myasthenic syndrome affecting limb-girdle muscles, a mutation in this gene was found. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Mar 2015] |
| UniProt Code: | O00468 |
| NCBI GenInfo Identifier: | 54873613 |
| NCBI Gene ID: | 375790 |
| NCBI Accession: | NP_940978.2 |
| UniProt Secondary Accession: | O00468,Q5SVA1, Q5SVA2, Q60FE1, Q7KYS8, Q8N4J5, Q96IC1 Q9BTD4, |
| UniProt Related Accession: | O00468 |
| Molecular Weight: | |
| NCBI Full Name: | agrin isoform 2 |
| NCBI Synonym Full Names: | agrin |
| NCBI Official Symbol: | AGRN |
| NCBI Official Synonym Symbols: | CMS8; AGRIN; CMSPPD |
| NCBI Protein Information: | agrin |
| UniProt Protein Name: | Agrin |
| Protein Family: | Agrin |
| UniProt Gene Name: | AGRN |
| 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. |
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