Description
Human CAF (c-terminal fragemnte of agrin) ELISA Kit
The Human CAF (C-terminal fragment of agrin) ELISA Kit is specifically designed for the accurate measurement of CAF levels in human samples including serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring dependable and consistent results, making it an ideal tool for various research purposes.CAF, also known as agrin, plays a crucial role in the development and maintenance of neuromuscular junctions and synapses.
Dysregulation of CAF levels has been associated with neurological disorders such as myasthenia gravis and Alzheimer's disease, making it a valuable biomarker for studying these conditions and exploring potential therapeutic interventions.Overall, the Human CAF ELISA Kit provides researchers with a reliable and accurate method for quantifying CAF levels, offering valuable insights into the role of CAF in health and disease.
| Product Name: | Human CAF (c-terminal fragemnte of agrin) ELISA Kit |
| Product Code: | HUFI04702 |
| Size: | 96 Assays |
| Alias: | CAF ELISA Kit |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human CAF (c-terminal fragemnte of agrin) concentrations in serum plasma and other biological fluids. |
| Sensitivity: | < 28.125pg/ml |
| Range: | 46.875-3000pg/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human CAF (c-terminal fragemnte of agrin) and the recovery rates were calculated by comparing the measured value to the expected amount of Human CAF (c-terminal fragemnte of agrin) in samples. Enquire for more information. |
| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CAF (c-terminal fragemnte of agrin) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information. |
| CV(%): | Intra-Assay: CV<8% Inter-Assay: CV<10% |
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| 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) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 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
| 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 |
*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.
| Step | Protocol |
| 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 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. |
