1-Methyladenosine (m1A) ELISA Kit

Catalog No. ABIN6938802

This Colorimetric ELISA kit is designed for the quantitative measurement of Bacteria 1-Methyladenosine (m1A).

Quick Overview for 1-Methyladenosine (m1A) ELISA Kit (ABIN6938802)

Target: 1-Methyladenosine (m1A)

Reactivity: Bacteria

Detection Method: Colorimetric

Application: ELISA

Sample Type: Cell Samples, Plasma, Serum, Tissue Samples, Urine

Product Details

Purpose: The 1-Methyladenosine (m1A) ELISA Kit is a competitive enzyme immunoassay developed for rapid detection and quantitation of 1-methyladenosine in urine, serum, or plasma samples.

Analytical Method: Quantitative

Sensitivity: 2 ng/mL

Characteristics: The 1-Methyladenosine (m1A) ELISA Kit is a competitive enzyme immunoassay developed for rapid detection and quantitation of 1-methyladenosine in urine, serum, or plasma samples. The quantity of 1-methyladenosine in unknown samples is determined by comparing its absorbance with that of a known 1-methyladenosine standard curve. The kit has a 1-methyladenosine detection sensitivity of approximately 2 ng/mL. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown samples.

Components:

1. 96-well Protein Binding Plate : One 96-well strip plate.
2. Anti-1-Methyladenosine Antibody : One 10 μL vial of anti-1- Methyladenosine.
3. Secondary Antibody, HRP Conjugate (1000X) : One 20 μL vial.
4. Assay Diluent : One 50 mL bottle.
5. 10X Wash Buffer : One 100 mL bottle.
6. Substrate Solution : One 12 mL amber bottle.
7. Stop Solution : One 12 mL bottle.
8. 1-Methyladenosine Standard : One 10 μL vial of 5 mg/mL 1- Methyladenosine in DMSO.

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Reagent Preparation:

• 1-Methyladenosine Conjugate Coated Plate: Dilute the proper amount of 1-Methyladenosine Conjugate 1:1000 in 1X PBS depending on the number of required assays. (Example: Add 5 μL of 4 1-Methyladenosine Conjugate stock tube to 4.995 mL 1X PBS to coat 48 wells). Add 100 μL of this 1-methyladenosine conjugate coating solution to each well and incubate overnight at 4 °C. Remove the 1-methyladenosine conjugate coating solution and wash once with 1X PBS. Blot plate on paper towels to remove excess fluid. Add 200 μL of Assay Diluent to each well and block for 1-2 hr at room temperature. Transfer the plate to 4 °C and remove the Assay Diluent immediately before use. Note: The 1-methyladenosine-conjugate coated wells are not stable and should be used within 24 hrs after coating. Only coat the number of wells to be used immediately.
• 1X Wash Buffer: Dilute the 10X Wash Buffer to 1X with deionized water. Stir to homogeneity.
• Anti-1-Methyladenosine Antibody and Secondary Antibody, HRP Conjugate (1000X): Immediately before use dilute the Anti-1-Methyladenosine Antibody 1:1000 and Secondary Antibody 1:1000 with Assay Diluent. Do not store diluted solutions.

Assay Procedure:

1. Prepare and mix all reagents thoroughly before use. Each 1-methyladenosine sample including unknown and standard should be assayed in duplicate.
2. Add 50 μL of unknown sample or 1-Methyladenosine Standard to the wells of the 1- Methyladenosine Conjugate Coated Plate. Incubate at room temperature for 10 minutes on an orbital shaker.
3. Add 50 μL of the diluted Anti-1-Methyladenosine Antibody to each well, incubate at room temperature for 1 hour on an orbital shaker.
4. Wash microwell strips 3 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
5. Add 100 μL of the diluted Secondary Antibody, HRP Conjugate to all wells.
6. Incubate at room temperature for 1 hour on an orbital shaker.
7. Wash microwell strips 3 times according to step 4 above. Proceed immediately to the next step.
8. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature on an orbital shaker. Actual incubation time may vary from 2-30 minutes. Note: Watch plate carefully, if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation.
9. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).
10. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length.

Restrictions: For Research Use only

Handling

Handling Advice: Avoid multiple freeze/thaw cycles.

Storage: -20 °C,-80 °C

Storage Comment: Upon receipt, aliquot and store the Anti-1-Methyladenosine Antibody and 1-Methyladenosine Standard at -20°C and the 1-Methyladenosine Conjugate (1000X) at -80°C to avoid multiple freeze/thaw cycles. Store all other components at 4°C.

Target Details

Target: 1-Methyladenosine (m1A)

Background: Modifications to ribonucleic acids (RNAs) are integrally involved in the function, integrity, metabolism, and biosynthesis of RNA molecules. There are over 100 RNA modifications that have been discovered with the majority occurring in transfer RNA (tRNA), but they can also be found in ribosomal RNA (rRNA), small nuclear RNA (snRNA), messenger (mRNA), and long non-coding RNA (ncRNA). Urinary excretion of modified nucleosides has been linked to a variety of cancers, AIDS, and other malignant disease states. 1-methyladenosine (m1A) is a prominent reversible post-transcriptional RNA modification that influences the function and structure of tRNA and rRNA. 1-Methyladenosine occurs at positions 9, 14, and 58 of tRNA, with position 58 (m1A58) being important to tRNA stabilization. Methyltransferases catalyze the addition of a methyl group to the first position nitrogen on the adenosine base structure, resulting in the addition of a positive charge to the molecule. The reaction is reversible with demethylases such as ALKBH3. 1-methyladenosine can affect ribosomal biogenesis, antibiotic resistance in bacteria, react to environmental stress in tRNA, and interfere with Watson-Crick base pairing, which ultimately affects reverse transcription and protein translation. Urine samples of cancer, rheumatoid arthritis, and AIDS patients have all demonstrated high levels of m1A, which supports its role as a functional detection biomarker. Elevated serum levels of 1- methyladenosine have been detected under stress conditions. Recent 1-methyladenosine research is uncovering new roles and mechanisms involving 1-methyladenosine. While m1dA in DNA is considered a form of damage leading to genomic mutations, and thus requiring repair, endogenous enzymes proliferate the m1A modification in RNA. The m1A at position 9 (m1A9) is known to stabilize the canonical cloverleaf motif in tRNA. In addition, the m1A58 of tRNA is present in all eukaryotic types which may imply it is vital to molecular structure and stability. While 1-methyladenosine has been identified as an early malignant disease marker, its complete role as a modified nucleoside has yet to be elucidated.