Description
Human CUBN (Cubilin) ELISA Kit
The Human CUBN (Cubilin) ELISA Kit offered by Assay Genie is a powerful tool for detecting levels of Cubilin in human biological samples such as serum, plasma, and cell culture supernatants. This kit is highly sensitive and specific, providing accurate and reproducible results for a variety of research applications.Cubilin is an essential protein involved in the process of receptor-mediated endocytosis, particularly in the uptake of important nutrients and vitamins. Dysregulation of Cubilin function has been linked to various health conditions, including metabolic disorders and kidney diseases, highlighting its importance as a biomarker for studying these conditions and developing potential therapeutic interventions.
With the Assay Genie Human CUBN (Cubilin) ELISA Kit, researchers can delve deeper into the role of Cubilin in physiological and pathological processes, paving the way for new insights and advancements in the field of biomedical research.
| Product Name: | Human CUBN (Cubilin) ELISA Kit |
| Product Code: | HUFI05335 |
| Size: | 96 Assays |
| Alias: | Cubn ELISA Kit, gp280 ELISA Kit, IFCR ELISA Kit, MGA1 ELISA Kit, cubilin ELISA Kit, intrinsic factor-cobalamin receptor ELISA Kit, gp280 ELISA Kit, IFCR ELISA Kit, MGA1 ELISA Kit |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human CUBN (Cubilin) concentrations in serum plasma and other biological fluids. |
| Sensitivity: | < 0.094ng/ml |
| Range: | 0.156-10ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human CUBN (Cubilin) and the recovery rates were calculated by comparing the measured value to the expected amount of Human CUBN (Cubilin) in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CUBN (Cubilin) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
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| 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: | CUBN: Cotransporter which plays a role in lipoprotein, vitamin and iron metabolism, by facilitating their uptake. Binds to ALB, MB, Kappa and lambda-light chains, TF, hemoglobin, GC, SCGB1A1, APOA1, high density lipoprotein, and the GIF-cobalamin complex. The binding of all ligands requires calcium. Serves as important transporter in several absorptive epithelia, including intestine, renal proximal tubules and embryonic yolk sac. Interaction with LRP2 mediates its trafficking throughout vesicles and facilitates the uptake of specific ligands like GC, hemoglobin, ALB, TF and SCGB1A1. Interaction with AMN controls its trafficking to the plasma membrane and facilitates endocytosis of ligands. May play an important role in the development of the peri-implantation embryo through internalization of APOA1 and cholesterol. Binds to LGALS3 at the maternal-fetal interface. Defects in CUBN are a cause of recessive hereditary megaloblastic anemia 1 (RH-MGA1); also known as MGA1 Norwegian type or Imerslund-Grasbeck syndrome (I-GS). RH-MGA1 is due to selective malabsorption of vitamin B12. Defects in vitamin B12 absorption lead to impaired function of thymidine synthase. As a consequence DNA synthesis is interrupted. Rapidly dividing cells involved in erythropoiesis are particularly affected. |
| UniProt Protein Details: | Chromosomal Location of Human Ortholog: 10p12.31 Cellular Component: Golgi apparatus; lysosomal lumen; lysosomal membrane; endoplasmic reticulum; brush border membrane; coated pit; cytosol; extrinsic to external side of plasma membrane; membrane; endocytic vesicle; apical plasma membrane; plasma membrane; endosome membrane Molecular Function:protein binding; protein homodimerization activity; transporter activity; receptor activity; calcium ion binding; cobalamin binding Biological Process: steroid metabolic process; receptor-mediated endocytosis; cholesterol metabolic process; vitamin metabolic process; cobalamin metabolic process; lipoprotein transport; cobalamin transport; tissue homeostasis; lipoprotein metabolic process; water-soluble vitamin metabolic process; vitamin D metabolic process Disease: Megaloblastic Anemia 1 |
| NCBI Summary: | Cubilin (CUBN) acts as a receptor for intrinsic factor-vitamin B12 complexes. The role of receptor is supported by the presence of 27 CUB domains. Cubulin is located within the epithelium of intestine and kidney. Mutations in CUBN may play a role in autosomal recessive megaloblastic anemia. [provided by RefSeq, Jul 2008] |
| UniProt Code: | O60494 |
| NCBI GenInfo Identifier: | 126091152 |
| NCBI Gene ID: | 8029 |
| NCBI Accession: | NP_001072.2 |
| UniProt Secondary Accession: | O60494,Q5VTA6, Q96RU9, B0YIZ4, |
| UniProt Related Accession: | O60494 |
| Molecular Weight: | 398,736 Da |
| NCBI Full Name: | cubilin |
| NCBI Synonym Full Names: | cubilin (intrinsic factor-cobalamin receptor) |
| NCBI Official Symbol: | CUBN |
| NCBI Official Synonym Symbols: | IFCR; MGA1; gp280 |
| NCBI Protein Information: | cubilin; 460 kDa receptor; cubilin precursor variant 1; cubilin precursor variant 2; intestinal intrinsic factor receptor; intrinsic factor-vitamin B12 receptor |
| UniProt Protein Name: | Cubilin |
| UniProt Synonym Protein Names: | 460 kDa receptor; Intestinal intrinsic factor receptor; Intrinsic factor-cobalamin receptor; Intrinsic factor-vitamin B12 receptor |
| Protein Family: | Cubilin |
| UniProt Gene Name: | CUBN |
| UniProt Entry Name: | CUBN_HUMAN |
*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. |
| Antibodies |
| CUBN Antibody (PACO05397) |
