Elastin is one of the extracellular matrix (ECM) proteins with the collagen that consists of many hydrophobic amino acids such as alanine, glycine, valine, and proline. Elastin is the elastic fi brous protein, which located in aorta, ligaments, lung, skin, and connective tissue. It is wrapped around the
coil-like collagen, to maintain the shape of the tissue and giving the elasticity-stretch.
Elastin is abundant in medial portion of the vessel wall, the content as arteriestake is thick and blood pressure will be higher. Elasticity and fl exibility acting on blood vessels are maintained by elastin.
Elastin in the vessel wall and skin tissue is also glycated by diseases such as aging and diabetes. It has been reportedto cause sclerotic change and aging of blood vessels and skin. Elastin Glycation Assay Kit provides rapid detection of fluorescent AGEs found in elastin glycated with glyceraldehyde and
screening of inhibitory eff ects of your samples. This kit provides suffi cient reagents to perform up to 192 assays. This kit is ideal for functional materials development that is focused such as lifestyle-related diseases and aging prevention
research for blood vessels and ligaments.
Principle: Elastin Glycation Assay Kit is a complete assay system designed to measure the fluorescent AGEs formed in elastin, when the elastin is glycated with glyceraldehyde. The fluorescent AGEs can be detected with the fluorescence microplate reader equippedwith a 370nm excitation filter and 440nm emission filter.
Protocol: (1) Elastin solution is stored at room temperature (18-25℃) before testing.
(2) Add 50 uL of Elastin solution to the 96-well black plate.
(3) Prepared Anti-glycation Standard (0.8 mM and 4mM) by diluting the 20 mM Aminoguanidine solution.
(4) Add 40 uL of the 0, 0.8, 4 and 20 mM Aminoguanidine (Anti-glycation Standard) or samples to each well.
Dissolve the samples with the dilution buff er and fi ltratewith 0.22 um.
(5) Add 10uL of 500 mM Glyceraldehyde Solution to each well. Mix thoroughly.
(6) Immediately begin reading standard and sample wells with a fl uorescent microplate reader with the
Excitation wavelength of 370 nm and an emissionwavelength of 440 nm by fl uorescence bottom reading.
Peg this fl uorescence intensity at before incubation (0 hr) and describe "Fluorescent intensity A".**
(7) Incubate the plate for 3-6 days at 37 ℃ under the high humidity condition to avoiddrying the well up
(but do not use CO2 incubator).
(8) Read the fl uorescent intensity after 3-6 days with a fl uorescent microplate reader at 37℃.
Peg the fl uorescence intensity at after incubation for 3-6 days fl uorescent intensity anddescribe
"Fluorescent intensity B".
(9) The reduction of fl uorescence intensity (Fluorescent intensity B － Fluorescent intensity A) from control
fluorescence intensity is the inhibitory eff ect of glycation.
** In case samples contain fl orescent material, subtract the fl orescence intensity of the sample group
without addition of glyceraldehyde (as "sample blank") from the group with glyceraldehyde.
