Description
Human MUC4 (Mucin 4) ELISA Kit
The Human MUC4 (Mucin-4) ELISA Kit is specifically designed for the accurate quantification of MUC4 levels in human serum, plasma, and cell culture supernatants. This kit boasts high sensitivity and specificity, ensuring precise and reproducible results for a variety of research purposes.MUC4 is a key mucin protein that plays a critical role in various cellular functions, including cell adhesion and signaling. Abnormal expression of MUC4 has been linked to cancer development and progression, as well as inflammatory and immune-related diseases.
As such, accurate measurement of MUC4 levels can provide valuable insights into disease mechanisms and potential therapeutic targets.Utilizing the Human MUC4 ELISA Kit allows researchers to easily and reliably quantify MUC4 levels in biological samples, facilitating a deeper understanding of its role in disease pathogenesis and progression. This kit is an essential tool for studies focusing on cancer biology, inflammation, and mucin-related disorders.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 0.31-20 ng/mL |
| Sensitivity: | 0.19 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human MUC4 in samples. No significant cross-reactivity or interference between Human MUC4 and analogues was observed. |
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human MUC4. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human MUC4 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human MUC4, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human MUC4. The concentration of Human MUC4 in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | MUC4: May play a role in tumor progression. Ability to promote tumor growth may be mainly due to repression of apoptosis as opposed to proliferation. Has anti-adhesive properties. Seems to alter cellular behavior through both anti-adhesive effects on cell-cell and cell-extracellular matrix interactions and in its ability to act as an intramembrane ligand for ERBB2. Plays an important role in cell proliferation and differentiation of epithelial cells by inducing specific phosphorylation of ERBB2. The MUC4-ERBB2 complex causes site-specific phosphorylation of the ERBB2 'Tyr-1248'. In polarized epithelilal cells segragates ERBB2 and other ERBB receptors and prevents ERBB2 from acting as a coreceptor. The interaction with ERBB2 leads to enhanced expression of CDKN1B. The formation of a MUC4-ERBB2-ERBB3-NRG1 complex leads to down-regulation of CDKN1B, resulting in repression of apoptosis and stimulation of proliferation. 14 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Membrane protein, integral Chromosomal Location of Human Ortholog: 3q29 Cellular Component: extracellular space; Golgi lumen; integral to plasma membrane; vesicle Molecular Function:ErbB-2 class receptor binding Biological Process: maintenance of gastrointestinal epithelium; O-glycan processing |
| NCBI Summary: | The major constituents of mucus, the viscous secretion that covers epithelial surfaces such as those in the trachea, colon, and cervix, are highly glycosylated proteins called mucins. These glycoproteins play important roles in the protection of the epithelial cells and have been implicated in epithelial renewal and differentiation. This gene encodes an integral membrane glycoprotein found on the cell surface, although secreted isoforms may exist. At least two dozen transcript variants of this gene have been found, although for many of them the full-length transcript has not been determined or they are found only in tumor tissues. This gene contains a region in the coding sequence which has a variable number (>100) of 48 nt tandem repeats. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q99102 |
| NCBI GenInfo Identifier: | 338817990 |
| NCBI Gene ID: | 4585 |
| NCBI Accession: | Q99102. 4 |
| UniProt Secondary Accession: | Q99102,O95938, Q9GZM2, Q9GZV6, Q9H481, Q9H482, Q9H483 Q9H484, Q9H485, Q9H486, Q9H487, Q9H4D6, |
| UniProt Related Accession: | Q99102 |
| Molecular Weight: | 206,605 Da |
| NCBI Full Name: | Mucin-4 |
| NCBI Synonym Full Names: | mucin 4, cell surface associated |
| NCBI Official Symbol: | MUC4 |
| NCBI Official Synonym Symbols: | ASGP; MUC-4; HSA276359 |
| NCBI Protein Information: | mucin-4 |
| UniProt Protein Name: | Mucin-4 |
| UniProt Synonym Protein Names: | Ascites sialoglycoprotein; ASGP |
| Protein Family: | Mucin |
| UniProt Gene Name: | MUC4 |
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (ng/mL) | O.D | Average | Corrected |
| 20 | 2.478 2.482 | 2.48 | 2.42 |
| 10 | 1.699 1.711 | 1.705 | 1.645 |
| 5 | 0.957 0.945 | 0.951 | 0.891 |
| 2.5 | 0.515 0.553 | 0.534 | 0.474 |
| 1.25 | 0.287 0.269 | 0.278 | 0.218 |
| 0.63 | 0.18 0.154 | 0.167 | 0.107 |
| 0.31 | 0.111 0.117 | 0.114 | 0.054 |
| 0 | 0.052 0.068 | 0.06 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human MUC4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human MUC4 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (ng/mL) | 1.00 | 2.00 | 9.80 | 1.00 | 2.00 | 9.10 |
| Standard deviation | 0.10 | 0.10 | 0.50 | 0.10 | 0.10 | 0.30 |
| C V (%) | 10.00 | 5.00 | 5.10 | 10.00 | 5.00 | 3.30 |
Recovery
The recovery of Human MUC4 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
| Serum (n=5) | 90-106 | 97 |
| EDTA plasma (n=5) | 90-105 | 96 |
| Cell culture media (n=5) | 92-104 | 97 |
Linearity
Samples were spiked with high concentrations of Human MUC4 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
| 1:2 | Range (%) | 90-107 | 92-104 | 90-105 |
| Average (%) | 97 | 97 | 96 | |
| 1:4 | Range (%) | 100-117 | 82-95 | 93-110 |
| Average (%) | 107 | 87 | 100 | |
| 1:8 | Range (%) | 94-109 | 83-96 | 95-112 |
| Average (%) | 102 | 88 | 103 | |
| 1:16 | Range (%) | 97-113 | 85-99 | 93-109 |
| Average (%) | 103 | 92 | 99 |
An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
| Micro ELISA Plate(Dismountable) | 8 wells ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4°C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- Aliquot 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
- Aspirate the liquid from each well, do not wash. Immediately add 100 µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37 °C.
- Aspirate or decant the solution from the plate and add 350 µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- Add 90 µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37 °C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
