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Home / Assay Kits / ELISA Kits

MAT Activity Assay 3

SKU : BHE16600031

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Arthus Biosystems

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Mfr.No.	IK00403
Description	Principle of the assay : This assay kit is designed to calculate MAT activity through measuring the concentration of the main product SAM. The chemical reaction above and the competition enzyme-linked immune-absorbent assay (cELISA) are carried out simultaneously, so that the SAM synthesized from the chemical reaction competes with antigens coated on the micro-titer plate for the horseradish peroxidase (HRP) -conjugated anti-SAM antibody the minute it is generated. The SAM is quantified based on the amount of the HRP-anti-SAM antibodies that was made not to bind to the micro-titer plate. Sample collection and storage : 1. Make sure samples do not contain SAM, NaN3 and insoluble substance to avoid pseudo or non-specific inhibition which contributes to the higher MAT activity incorrectly or interfere with HRP. 2. In case of much highly purified MAT with higher activity, the amount of SAM generate may exceed the detection range of this kit. Appropriate sample dilution with provided Sample Diluent is required before testing in this situation. Data processing: Always clear the background by subtracting the absorbance at 450 nm (0D450) of blank well from that of the test well. The binding rate of each well (standard or sample) is equal to A/ASO, with A being the average absorbance of the standard wells or the sample wells, ASObeing the average absorbance of the SO standard wells. Creation of standard curve: Construct a standard curve by plotting the binding rate of each standard on the y-axis against the logarithm of its concentration on x-axis. Use quadratic polynomial curve to fit the data (r>0.99). The SAM level of the sample can be calculated by substituting its binding rate into the standard curve equation. Then multiply the extent of dilution if the sample has been diluted Definition of MAT activity: The amount of MAT required to produce 1 nM under 37 °C per minute is defined as a unit of enzyme activity (U) Specific Activity= Enzyme activity (U) / Amount of enzyme (mg) Procedure: 1. Warm up all reagents by putting them to room temperature and mix them well. Take out an appropriate number of wells and put the remaining wells back into the original sealing bag. Seal and store it properly. 2. Add 30ul sample diluent (to blank well), standards into each well. For the sample wells, add 10ul MAT Substrate, then add 20ul test sample. If the test sample needs to be diluted, add after diluting it in another tube with provided Sample Diluent. Duplicate wells are recommended. 3. Prepare HRP-antibody solution: Dilute HRP-antibody with HRP-antibody Diluent at 1 :300, which should be used up within a week. Mix thoroughly and store in dark. Prepare the right amount that will be used in a week. Note: Always thoroughly centrifuge the HRP-antibody vial to recover all the 15-17ul HRP-antibody. It is recommended to collect all samples and measure them together in 1-3 experiments in a week by using 4.5ml of HRP-antibody Diluent to recover all HRP-antibody from the vial by washing it out several times. 4. Add ?Oul HRP-antibody prepared from the previous step into each well except for the blank well. Notice: If the MAT activity of the sample is low or hard to detect, allow the sample and the MAT Substrate from Step 2 to react first as described in Step 5 below at 37°C for 20min. Then add ?Oul HRP-antibody followed by incubating the plate at 37°C for about 40min. If you are not concerned about potential low MAT activity from the sample, add the ?Oul HRP-antibody directly and proceed to the next step. 5. Put the plate onto oscillator and shake for a while to mix the reagents, and seal the plate with micro-plate sealer. Incubate the plate at 37°C for 1 hour. 6. Peel the sealer carefully, and discard the remaining solution in the wells. Add at least 300ul wash solution in each well and maintain this state for 30 seconds, then remove wash solution. Repeat these steps 3 times to finish washing process. Or use auto-washer instead. 7. Add HRP substrate TMB and add 100ul blending substrate to each well. Shake gently and seal the plate. Incubate the plate at 37°C for 15 minutes in dark. 8. Add 50ul Stop Solution to end reaction. 9. Measure absorbance of each well at 450nm within 15 minutes. Set zero according to the blank well. 10. MAT Positive Control is used to qualitatively evaluate MAT Substrate. Two-test supply can be used in 3 tests after dilution if necessary
Notes	1. Using reagents and samples without warm-up or ambient temperature less than 20°C may lead to reduced 0D450 reading 2. Extra drying of wells after washing may have negative effects on the results, such as poor standard curve and poor repeatability. To avoid it, perform the next step immediately after washing 3. Mix solution well and wash completely, as these procedures will have influence on the assay. 4. Seal the plate with micro-plate sealer to avoid light. The sealers should be disposed after each use to avoid cross contaminations. 5. Duplicate wells for standards and samples are recommended, and quadratic polynomial is suggested for standard curve fitting (R2>0.99) The detected concentration of quality control vial should be in the detection range. 6. If the absorbance of sample well was lower than that of S7 standard, please dilute the sample at least 10-fold and test it again until the sample can be properly detected 7. The concentrated wash solution may form crystals. Warm it up to allow salts to dissolve completely before diluting 8. In a rare case when MAT Positive Control failed to show SAM synthesis due to reduced MAT activity, your test may still work as the MAT substrates are more stable and are still good 9. The HRP substrate should be kept in dark 10. The Stop Solution is diluted sulfuric acid. Avoid direct contact with skin and other things

Target	Except for parasites that rely on host for living, cells from all organisms have methionine adenyltransferase (MAT, EC2.5.1.6), also known as Sadenosylmethionine synthetase. MAT genes have been found to be exceptionally conserved throughout evolution. It

Storage	Storage condition: Except for the HRP substrate and stop solution that are stored at 2-8°C, all other ingredients and strips can be frozen stored. Expiration: About 6 months from the date of shipment when storing it in refrigerator. If not planning to use

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SKU	Supplier No.	Size	Your price	Quantity
BHE16600031	IK00403	48 tests	$449		Add to Cart

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