i am doing alpha- amylase inhibition assay for an in vitro gastrointestinal digest of an ayurvedic formulation- both pepsin digest and pancreatic digest. but for both of digest, amylase activity's absorbance is higher than that of my negatgive control, which shows 100% Amylase activity.
my protocol goes like this:
· 100µl of the sample to be taken in a 96-well plate.
· It is then serially diluted with 50µl freshly prepared 0.02M NaP buffer
· To these wells added, 50µl of 0.5µl/ml concentration amylase dissolved in 0.02M NaP buffer solution (pH- 6.9)
· Incubated for 30mins in room temperature
· Added 50µl of freshly prepared 1% starch prepared using 0.02M NaP buffer
· After 10mins of incubation under room temperature, added freshly prepared DNS solution.
· Kept the well in a boiling water bath for 8 mins
· Colour change was observed from bright yellow to bright orange colour.
· This orange color solution was measured at 540-550 nm To check the activity of amylase in the given sample.
BUT when I read other papers related to the same assay, ppl have cooled down the solution to room temperature and diluted the solution using water. i am not able to understand this step why it is so??
Dilute your sample and the calculate using dilution factor. Dilution is neede as your sample might have higher amounts so colour must be very dark.
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