Mucin 1, Cell Surface Associated (MUC1) ELISA Kit

Antigen: Mucin 1, Cell Surface Associated (MUC1)

Synonyms: EMA, PEM, PUM, KL-6, MAM6, PEMT, CD227, H23AG, MGC188069, MUC1

Reactivity: Rabbit

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN577593

Additional Information

Alternative name: Mucin 1 (MUC1)

Sample Type: Serum, Plasma

Specificity: This assay recognizes recombinant and natural rabbit MUC1. No significant cross-reactivity or interference was observed.

Sensitivity: The minimum detectable dose of rabbit MUC1 is typically less than 0.53 pg/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. Detection Range: The minimum detectable dose of rabbit MUC1 is typically less than 0.53 pg/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with rabbit MUC1. Standards or samples are then added to the appropriate microtiter plate wells with Horseradish Peroxidase (HRP) -conjugated antibody preparation specific for rabbit MUC1 ， mix well and incubated. The more the amount of MUC1 in samples, the less Horseradish Peroxidase (HRP) -conjugated antibody preparation specific for rabbit MUC1 bound by pre-coated rabbit MUC1. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. And the color develops in opposite to the amount of rabbit MUC1 in the sample. The color development is stopped and the intensity of the color is measured.

Protocol: Reagent Preparation:
Bring all reagents to room temperature before use. 1. Wash Buffer If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
Assay Procedure:
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well. 2. Recommend to dilute the serum samples with Sample Diluent(1:4000) before test. The suggested 4000-fold dilution can be achieved by adding 5 μl sampl e to 195 μl of Sample Diluent first, then complete the 4000-fold dilution by adding 5 μl of this solution to 495 μl of Sample Diluent. The recommended dilution factor is for reference only. The optimal dilution factor should be determined by users according to their particular experiments. 3. Set a Blank well without any solution , 50μl Standard or Sample to other wells. 6 4. Add 50 μ l HRP-Conjugate immediately after adding each sample ( Don’t to Blank well!). Mix well with the pipette or shake the plate gently for 60 seconds. 5. Then incubate for 30 minutes at 37°C. 6. Aspirate each well and wash, repeating the process five times for a total of five washes. Wash: Fill each well with Wash Buffer (200μl) and let it stand for one minute, then remove the liquid by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel. Complete removal of liquid at each step is essential to good performance. 7. Add 90 μl of TMB Substrate to each well. Incubate for 20 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 8. Add 50 μl of Stop Solution to each well when the last four wells containing the lowest concentration of standards develop obvious blue color. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 9. Determine the optical density of each well within 15 minutes, using a microplate reader set to 450 nm.
Calculation of Results:
Using the professional soft ",Curve Exert 1.3", to make a standard curve is recommended, which can be downloaded from our web. 7 Average the duplicate readings for each standard, control, and sample and divide the average zero standard optical density. Create a standard curve by reducing the data using computer software. As an alternative, construct a standard curve by plotting the absorbance ratio for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the MUC1concentrations versus the ratio and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Limitations of the Procedure:
The kit should not be used beyond the expiration date on the kit label.Do not mix or substitute reagents with those from other lots or sources.Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded. 8.
Sample collection and storage:
Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediately or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay.

Assay Procedure: Assay Time: 1-3h
Sample Volume: 50-100ul

Application Notes: Detection Wavelength: 450 nm

Components: Reagent (Quantity): Assay plate (1), Standard 5x1ml HRP-conjugate (1x6ml), Sample Diluent 4x20ml Wash Buffer (25×concentrate) (1x20ml), TMB Substrate (1x10ml), Stop Solution (1x10ml)

Material not included: Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer. An incubator which can provide stable incubation conditions up to 37°C±0.5°C.

Storage: 1. Unopened test kits should be stored at 2-8 C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit, provided it is stored as prescribed above. Refer to the package label for the expiration date. 2. Opened test plate should be stored at 2-8 C in the aluminum foil bag with desiccants to minimize exposure to damp air. The kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Restrictions: For Research Use only