Methylglyoxal (MG) ELISA 试剂盒

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实验类型
Competition ELISA
应用范围
ELISA
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原理 First, an MG conjugate is coated on the ELISA plate. The unknown MG protein samples or MG- BSA standards are then added to the MG conjugate preabsorbed plate. After a brief incubation, the anti-MG antibody is added, followed by an HRP conjugated secondary antibody. The content of MG protein adducts in unknown samples is determined by comparison with the predetermined MG-BSA standard curve.
品牌 OxiSelect™
样品类型 Serum, Plasma
Analytical Method Quantitative
检测方法 Colorimetric
产品特性 The OxiSelect™ Methylglyoxal (MG) ELISA Kit is an enzyme immunoassay developed for rapid detection and quantitation of MG-H1 (methyl-glyoxal-hydro-imidazolone) protein adducts. The quantity of MG adduct in protein samples is determined by comparing its absorbance with that of a known MG-BSA standard curve. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown protein samples.
组件
  1. 96-well Protein Binding Plate : One strip well 96-well plate
  2. Anti-MG Antibody (1000X) : One 10 μL vial of anti-MG antibody
  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 (Part. No. 310808): One 12 mL bottle

Box 2 (shipped on blue ice packs)

试剂未包括
  1. Protein samples such as purified protein, plasma, serum, cell lysate
  2. 1X PBS
  3. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
  4. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
  5. Multichannel micropipette reservoir
  6. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)
背景 The non-enzymatic reaction of reducing carbohydrates with lysine side chains and N-terminal amino groups of macromolecules (proteins, phospholipids and nucleic acids) is called the Maillard reaction or glycation. The products of this process, termed advanced glycation end products (AGEs), adversely affect the functional properties of proteins, lipids and DNA. Tissue levels of AGE increase with age and the formation of AGEs is predominantly endogenous, though these products can also be derived from exogenous sources such as food and tobacco smoke. AGE modification of proteins can contribute to the pathophysiology of aging and long-term complications of diabetes, atherosclerosis and renal failure. AGEs also interact with a variety of cell-surface AGE-binding receptors (RAGE), leading either to their endocytosis and degradation or to cellular activation and pro-oxidant or pro- inflammatory events. Several AGE structures have been reported, such as Nε-(carboxymethyl) lysine (CML), Nε- (carboxyethyl) lysine (CEL), pentosidine, and Methylglyoxal (MG) derivatives. MG is formed through non-oxidative mechanisms from triose phosphates during anaerobic glycolysis and it can modify amino acids, nucleic acids, and proteins. MG reacts with arginine, lysine and cysteine residues of proteins to form AGEs. MG is involved in various pathological processes. For example, MG derivatives are found elevated in diabetes.
说明

  • Rapid detection and quantitation of MG-H1 (methyl-glyoxal-hydro-imidazolone) protein adducts
  • Provides sufficient reagents to perform up to 96 assays, including standard curve and unknown protein samples

板类型 Uncoated
试剂准备
  • MG Conjugate Coated Plate: Note: The MG 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. 1. Immediately before use, prepare 1X Conjugate Diluent by diluting the 100X Conjugate Diluent in 1X PBS. Example: Add 50 μL to 4.95 mL of 1X PBS. 3 2. Immediately before use, prepare 500 ng/mL of MG Conjugate by diluting the 1.0 mg/mL MG Conjugate in 1X Conjugate Diluent in two step dilutions. Example: Add 5 μL of 1.0 mg/mL MG Conjugate to 995 μL of 1X PBS, vortex thoroughly, and transfer 500 μL to another tube containing 4.5 mL of 1X Conjugate Diluent. 3. Add 100 μL of the 500 ng/mL MG Conjugate to each well to be tested and incubate overnight at 4 °C. Remove the MG Conjugate coating solution and wash twice 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 hr at room temperature on an orbital shaker. Transfer the plate to 4 °C and remove the Assay Diluent immediately before use.
  • 1X Wash Buffer: Dilute the 10X Wash Buffer to 1X with deionized water. Stir to homogeneity.
  • Anti-MG Antibody and Secondary Antibody: Immediately before use, dilute the Anti-MG antibody 1:1000 and Secondary Antibody 1:1000 with Assay Diluent. Do not store diluted solutions.
实验流程
  1. Prepare and mix all reagents thoroughly before use. Each MG sample including unknown and standard should be assayed in duplicate.
  2. Add 50 μL of unknown sample or MG-BSA standard to the wells of the MG Conjugate coated plate. If needed, unknown samples may be diluted in 1X PBS containing 0.1 % BSA before adding. Incubate at room temperature for 10 minutes on an orbital shaker. 4
  3. Add 50 μL of the diluted anti-MG antibody to each well, incubate at room temperature for 1 hour on an orbital shaker.
  4. Wash 3 times with 250 μL of 1X Wash Buffer 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 and incubate for 1 hour at room temperature on an orbital shaker. Wash the strip wells 5 times according to step 4 above.
  6. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well. Incubate at room temperature for 2-20 minutes on an orbital shaker. Note: Watch plate carefully, if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation.
  7. Stop the enzyme reaction by adding 100 μL of Stop Solution to each well. Results should be read immediately (color will fade over time).
  8. Read absorbance of each well on a microplate reader using 450 nm as the primary wave length.
限制 仅限研究用
注意事项 Avoid multiple freeze/thaw cycles.
储存条件 4 °C/-20 °C
储存方法 Upon receipt, aliquot and store the Anti-MG Antibody, MG-BSA Standard, MG Conjugate and 100X Conjugate Diluent at -20°C to avoid multiple freeze/thaw cycles. Store all other kit components at 4°C.
有引用在: Nishimoto, Koike, Inoue, Suzuki, Ogasawara: "Activation of Nrf2 attenuates carbonyl stress induced by methylglyoxal in human neuroblastoma cells: Increase in GSH levels is a critical event for the detoxification mechanism." in: Biochemical and biophysical research communications, Vol. 483, Issue 2, pp. 874-879, 2017 (PubMed).

Ueda, Zhao, Kim, Sparrow: "Photodegradation of retinal bisretinoids in mouse models and implications for macular degeneration." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 113, Issue 25, pp. 6904-9, 2016 (PubMed).

Morgan, Sheahan, Davies: "Perturbation of human coronary artery endothelial cell redox state and NADPH generation by methylglyoxal." in: PLoS ONE, Vol. 9, Issue 1, pp. e86564, 2014 (PubMed).

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