Columns A and B are the standard curve. The formula line was generated according to the standard curve and individual samples can be quantified with this graph.
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Abeta is a derivative of APP (Amyloid-beta Precursor Protein) processing, which is the major component of amyloid plaque in Alzheimer’s Disease (AD). Abeta 40/42 is produced via beta- and gamma-cleavage from APP; most of them will be secreted extracellularly. We used the Abeta 40/42 Human ELISA Kit was used to measure the Abeta 40/42 in tissue and cell culture medium. The Abeta42 Human ELISA Kit quantifies natural and recombinant Abeta 42 selectively in tissue culture medium, tissue homogenate, cerebrospinal fluid, and other samples.
ELISA
In our lab, we generated APP stable cell lines in HEK293. For example, if we plan to detect the effect of gamma-secretase on Aß generation, presenilins (the catalaytic component of gamma-secretase) will be transiently transfected into HEK293 with APP stably expressed. After 24h of transfection (in a 35mm plate), we change 1ml fresh medium and collect medium after another 24h, with the addition of a serine proteinease inhibitor.
The general steps are as follows: 1) make an Aß standard, 2) dilute the sample with dilution buffer, 3) add 50ul/well of the standard and samples to each well of a 96-plate; add 50ul/well primary Ab, 4) shake at room temperature for 3h, 5) wash and add secondary Ab and incubate at room temperature for 1h, 6) Wash and add chromogenic agent and incubate for 30min, 7) add stop solution and detect colorimetric development at 450nm.
It is very important to make sure your sample OD value is within the range of the standard curve.
The graph below shows the standard curve generated in a 96-well assay.
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