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Home / Assay Kits / ELISA Kits

SAM ELISA 2

SKU : BHE16600024

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Arthus Biosystems

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Mfr.No.	IK00202
Description	Principle of the assay : This direct competitive ELISA (Enzyme Linked lmmunosorbent assay) is designed to measure the level of S-adenosyl-L-methionine (SAM) in the sample. SAM conjugated with macromolecule is immobilized on the micro-titer plate. Standards and samples are pipetted into the wells, and then the HRP-conjugated antibody against SAM is added. The free SAM molecule in samples or standards competes with the immobilized SAM on the micro-titer plate surface for binding sites of the antibody. After discarding the mixed solution and washing each well, TMB substrate solution is added. The substrate solution turns blue under the effect of HRP (horseradish peroxidase), and changes into yellow once stop solution (acid) is added. The color develops in inverse proportion to the amount of SAM in the sample (or standards). The optical density of the remaining solution (0D450) is measured at 450nm using micro-plate spectrophotometer. The level of SAM in samples can be calculated through standard curve generated with standards. Sample collection and storage : 1. Sample collection must be carried out at 4 °C. Remove precipitation by centrifugation when necessary, and test sample as soon as possible or store at -20°C or below. Avoid repeated freeze-thaw cycles. 2. Avoid NaN3 in samples, since it will inactivate Horse radish peroxidase (HRP). 3. Some unknown factors may have effects on the assay (matrix effects). Dilute samples with sample diluent or prepare standards/samples with the same matrix as the actual sample matrix, which should avoid or reduce the matrix effects. Data processing: Always clear the background by subtracting the absorbance at 450 nm (0D450) of blank well from that of the test well. The binging 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, ASO being 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. Procedure: 1. Rewarm all reagents to room temperature and mix them well. Take out appropriate number of wells and put the remaining wells back into the Ziploc. Seal the Ziploc and stored it at -20°C. 2. Add 30ul sample diluent (blank well), standards and sample into each well. 3. Prepare HRP-conjugated antibody solution: Dilute HRP-antibody with HRP-antibody diluent at 1 :600, which should be used up within a week. If it will be used after a week, please prepare it when needed. Mix thoroughly and store in dark 4. Add 70ul HRP-conjugated antibody into each well except for the blank well. 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 TMB Substrate and add 100ul blending substrate to each well. Shake gently and seal the plate. Incubate the plate at 37°C for 15 minutes without light 8. Add 50ul Stop Solution to end reaction. 9. Measure absorbance of each well at 450 nm wavelength within 15 minutes. Set zero according to the blank well.
Notes	1. Using reagents and samples without rewarming or ambient temperature is less than 20°C may lead to reduced 0D450 values. 2. Extra drying post washing may have negative effect on the results, such as poor standard curve and repeatability. To avoid this, operate 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 and avoid light when incubating the plate. 5. Duplicate wells for standards and samples are recommended, and quadratic polynomial is suggested for standard curve fitting (r>0.99). The detected concentration of quality control vial should be in the detection range 6. The absorbance of sample well may be higher than that of SO standard well due to matrix effects. Dilute the sample at least ten-fold under this circumstance. If SAM level cannot be detected after 10-fold dilution, use the same matrix as the sample to be measured to prepare a new set of standards and the measure again. 7. The concentrated wash solution may crystallize. Warm it up to allow salts dissolved completely before diluting. 8. The micro-plate sealers should be disposable in order to avoid cross contamination. 9. The Substrate should be kept out of light. 10. The Stop Solution is diluted sulfuric acid. Avoid direct contact with skin and other things.

Target	Methionine is an amino acid with sulfur methyl group, which is converted into S-adenosylmethionine (SAM) with ATP by methionine adenosyltransferase (EC 2.5.1.6). SAM is the sole methyl group donor for over 50 kinds of biologically active substances such a

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
BHE16600024	IK00202	96 tests	$599		Add to Cart

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