Description
Human Melanoma-associated antigen 1 (MAGEA1) ELISA Kit
The Human Melanoma-Associated Antigen 1 (MAGEA1) ELISA Kit is specifically designed for the accurate detection of MAGEA1 levels in human serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring reliable and reproducible results for a variety of research applications.MAGEA1 is a known tumor antigen that is overexpressed in melanoma, making it a valuable biomarker for studying this aggressive form of skin cancer.
Detection of MAGEA1 levels can aid in the diagnosis and monitoring of melanoma progression, as well as in the development of potential targeted therapies for this disease.Overall, the Human Melanoma-Associated Antigen 1 (MAGEA1) ELISA Kit provides researchers with a valuable tool for studying melanoma and developing new approaches for its treatment.
| Product Name: | Human Melanoma-associated antigen 1 (MAGEA1) ELISA Kit |
| SKU: | HUEB1398 |
| Size: | 96T |
| Target: | Human Melanoma-associated antigen 1 (MAGEA1) |
| Synonyms: | Antigen MZ2-E, Cancer/testis antigen 1.1, MAGE-1 antigen, CT1.1, MAGE1, MAGE1A |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.625-40ng/mL |
| Sensitivity: | 0.326ng/mL |
| Intra CV: | 5.9% | ||||||||||||||||||||
| Inter CV: | 8.7% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | May be involved in transcriptional regulation through interaction with SNW1 and recruiting histone deactelyase HDAC1. May inhibit notch intracellular domain (NICD) transactivation. May play a role in embryonal development and tumor transformation or aspects of tumor progression. Antigen recognized on a melanoma by autologous cytolytic T-lymphocytes. |
| Uniprot: | P43355 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Melanoma-associated antigen 1 |
| Sub Unit: | Interacts with SNW1 and HDCA1. |
| Research Area: | Cancer |
| Subcellular Location: | Cytoplasm Nucleus |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | MAGE-A1: a tumor antigen expressed in a variety of tumor cells and in the testis. May play a role in embryonal development and tumor transformation or aspects of tumor progression. Antigen recognized on a melanoma by autologous cytolytic T-lymphocytes. |
| UniProt Protein Details: | Protein type:Cell surface Chromosomal Location of Human Ortholog: Xq28 Cellular Component: cytoplasm; plasma membrane; nucleus Molecular Function:protein binding; histone deacetylase binding Biological Process: transcription, DNA-dependent; negative regulation of Notch signaling pathway; negative regulation of transcription from RNA polymerase II promoter |
| NCBI Summary: | This gene is a member of the MAGEA gene family. The members of this family encode proteins with 50 to 80% sequence identity to each other. The promoters and first exons of the MAGEA genes show considerable variability, suggesting that the existence of this gene family enables the same function to be expressed under different transcriptional controls. The MAGEA genes are clustered at chromosomal location Xq28. They have been implicated in some hereditary disorders, such as dyskeratosis congenita. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P43355 |
| NCBI GenInfo Identifier: | 1170855 |
| NCBI Gene ID: | 4100 |
| NCBI Accession: | P43355.1 |
| UniProt Secondary Accession: | P43355,O00346, B2RC81, |
| UniProt Related Accession: | P43355,AAB33086 |
| Molecular Weight: | 309 |
| NCBI Full Name: | Melanoma-associated antigen 1 |
| NCBI Synonym Full Names: | melanoma antigen family A, 1 (directs expression of antigen MZ2-E) |
| NCBI Official Symbol: | MAGEA1 |
| NCBI Official Synonym Symbols: | CT1.1; MAGE1 |
| NCBI Protein Information: | melanoma-associated antigen 1; antigen MZ2-E; MAGE-1 antigen; melanoma antigen MAGE-1; cancer/testis antigen 1.1; melanoma antigen family A 1; melanoma-associated antigen MZ2-E; cancer/testis antigen family 1, member 1 |
| UniProt Protein Name: | Melanoma-associated antigen 1 |
| UniProt Synonym Protein Names: | Antigen MZ2-E; Cancer/testis antigen 1.1; CT1.1; MAGE-1 antigen |
| Protein Family: | Melanoma-associated antigen |
| UniProt Gene Name: | MAGEA1 |
| UniProt Entry Name: | MAGA1_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. |
