Team:Heidelberg/Notebook/Material Methods
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Laura Nadine (Talk | contribs) (→ELISA) |
Laura Nadine (Talk | contribs) (→Procedure) |
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#* incubate 1 hour at 37°C | #* incubate 1 hour at 37°C | ||
#* wash five times | #* wash five times | ||
- | + | #* add 100 µl substrate per well | |
- | #* add | + | |
- | + | ||
#* incubate at room temperature for 5 - 10 min | #* incubate at room temperature for 5 - 10 min | ||
- | #* stop with | + | #* stop with 100µl 2M H<sub>2</sub>SO<sub>4</sub> per well |
- | #* read at | + | #* read absorbance at 450 nm wavelength |
+ | |||
+ | |||
+ | ==== Buffers ==== | ||
+ | *'''Coating Buffer''' (0.68 g sodium carbonate, 3.675 g sodium bicarbonate, pH 9.2 with 5N HCl, ad 500 ml ddH<sub>2</sub>O) | ||
+ | |||
+ | *'''Diluting/Blocking Buffer (db)''' (0.25 ml Tween 20, 30 g BSA, add 1x PBS to 500 ml) | ||
+ | |||
+ | *'''Wash Buffer''' (0.5 ml Tween 20, 1L PBS 1x) | ||
+ | |||
+ | |||
+ | :'''Calibration''' | ||
+ | * 960 µl db + 40 µl pooled plasma = 200 ng/ml | ||
+ | * add 500 µl of above into 500 µl of db = 100 ng/ml | ||
+ | * serial dilutions continued down to 1.5625 ng/ml | ||
+ | |||
+ | |||
+ | :'''Detection reagents''' | ||
+ | * TMB Substrate Kit (Pierce, Thermo Scientific) | ||
+ | * 2M H<sub>2</sub>SO<sub>4</sub> | ||
=== Microscopy === | === Microscopy === |
Revision as of 17:28, 24 October 2010
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