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Description
Human METTL3 ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. 2. Plasma: Collect specimens using EDTA or heparin as an anticoagulant and centrifuge at 1000×g for 15 minutes at 2-8°C within 30 minutes of collection. Remove the supernatant for testing, or store at -20°C or -80°C, but avoid repeated freezing and thawing. 3. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for testing, or store at -20°C or -80°C, but avoid repeated freezing and thawing. Pre-Test Preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare a gradient standard working solution: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 20 ng/mL). Then dilute to the following concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 20 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of biotinylated detection antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10uL concentrate + 990uL universal diluent). Prepare and use immediately. 4. Prepare the enzyme conjugate working solution: 15 minutes before use, centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100× concentrated HRP enzyme conjugate to a 1× working concentration with universal diluent (e.g., 10 μL of concentrate + 990 μL of universal diluent). Prepare immediately. 5. Prepare the 1× wash solution: Dispense 10 mL of 20× wash solution into 190 mL of distilled water (concentrated wash solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing). Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return to 4°C. 2. Sample addition: Add 100 μL of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of universal diluent to the blank wells. Cover with a film and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the ELISA plate. This will reduce the impact of matrix effects on the test results. The sample concentration should be multiplied by the corresponding dilution factor when calculating the final sample concentration. It is recommended to run replicates for all test samples and standards.) 3. Add Biotinylated Antibody: Remove the ELISA plate and discard the liquid without washing. Add 100 μL of Biotinylated Antibody Working Solution directly to each well. Cover with a film and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid and add 300 μL of 1x Wash Solution to each well. Let stand for 1 minute, shake off the wash solution, and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Add Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a film and incubate at 37°C for 30 minutes. 6. Washing: Discard the liquid and wash the plate five times as in step 4. 7. Adding substrate: Add 90 μL of substrate (TMB) to each well, cover with a sealing film, and incubate at 37°C in the dark for 15 minutes. 8. Adding stop solution: Remove the ELISA plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450 nm. Calculating experimental results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as a correction factor. Plot the standard curve of the four-parameter logistic function on double-logarithmic graph paper, with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is higher than the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor. |
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| Theory | This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a capture antibody against methyltransferase-like protein 3 (METTL3). After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and to yellow by acid. The intensity of the color is positively correlated with the amount of methyltransferase-like protein 3 (METTL3) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Methyltransferase-like protein 3 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Methyltransferase-like protein 3 (METTL3) is an enzyme encoded by the METTL3 gene. This gene encodes the 70 kDa subunit of MT-A, which is part of the N6-adenosine methyltransferase enzyme. This enzyme is involved in the posttranscriptional methylation of internal adenosine residues in eukaryotic mRNA to form N6-methyladenosine (m6A). | |||||||||||||||||||||||||||||||||
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use. 2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out during incubation. 3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value. 4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue. 5. Avoid cross-contamination of reagents and specimens to prevent erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching agent will destroy the biological activity of the reagents in the kit. 8. Do not use expired products, and do not mix components with different product numbers and batches. 9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and samples and testing devices should be handled according to prescribed procedures. |
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| Storage Temp. | If the unopened kit is stored at 4°C, the shelf life is 6 months. | |||||||||||||||||||||||||||||||||
| Test Range | 0.312-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, cell culture supernatant |
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4.8 ★★★★★
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Product Reviews
★★★★★ 5
Great product
Style: SPF 50, Size: 6 Fl Oz (Pack of 2)
Product is great with good coverage and ease of use. Much better than comparable products because it is very light feeling and doesn't leave a heavy or sticky residue on your skin. Perfect protection for anyone who burns easily or fair skinned.
However I paid for the extra rushed 3 day shipping and it was delayed and I wasn't reimbursed the extra shipping charges even though it arrived 6 days later.
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Reviewed in the United States on February 16, 2024
★★★★★ 1
Save your money- don’t buy
Style: SPF 50, Size: 6 Fl Oz (Pack of 2)
Purchased this for our trip to Mexico. At two cruise ports we were doing a combination snorkel and pool day. I got this sunscreen because it was reef friendly and came with two bottles. Both bottles were IMPOSSIBLE to spray. You could not sunscreen yourself because you had to hold the bottle just right, push down with extreme force, and have your fingers place very specifically on the lid. My 13 year old and husband couldn’t even use the bottles. I did figure it out so I had to continually sunscreen everyone in the family. No one in my family has sensitive skin, but we all ended up with a fine rash after using this sunscreen. On days prior we used another brand and no one had a problem. We also reapplied often (every 60-90 min) and we still got pretty burnt. Maybe we got a bad batch…
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Reviewed in the United States on April 2, 2023
★★★★★ 5
All I use…from NB to 5!
Have used this brand for years, for many children, in sizes from NB to 5! Have used other “natural/organic” brands, but come back to these.
They are softer than 7th Gen and more absorbent. Also more absorbent than Honest Co. Earth’s Best also had great diapers, but are no longer available. I like that Babyganics has added the “wetness detector strip,” as it makes it handy for quick checks. The price is middle of the road, as far as natural/organic/chlorine free diapers go. The NB option is wonderful, as other brands (like Bambo Nature and Eco by Naty) start at 1s, which are too big for newborns.
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Reviewed in the United States on December 29, 2022
★★★★★ 4
Great ingredients, flawed product
I’ve been using these because they are TCF (total chlorine free) but unfortunately the tabs are hard to open on the side and tend to rip off easily…wasting a new, clean diaper. i’m contemplating switching brands :(
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Reviewed in the United States on September 19, 2024
★★★★★ 3
Janky
These are very cheaply made and barely stay on. The clasp to keep the diapers closed breaks half of the time when I'm trying to put a fresh diaper on. Won't be buying these again.
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Reviewed in the United States on January 30, 2023