I am performing in vitro anti inflammatory works. From different articles, I found out procedures which are having variations relate to reagents in each paper.
The methods you choose will depend upon what equipment you have available. Do you have a spectrophotometer, spectrofluorometer, plate reader, RT-PCR instrument, differential scanning calorimeter, SDS-PAGE? Do you need to do temperature ramping experiments?
As rightly said by Adam, the available methods are always varying with each other due to various reason, though they follows similar principle.
Select the methods always based on the facility you are having. Slight modification is from the original is accepted but it should not deviate the original principle of analysis.
Always standardize the methodology with known samples before going test samples, though you are following a reference.
Protease inhibition, in particular trypsin inhibitor was assayed by determining residual trypsin activity using Kakade et al. (1974) method with slight modification. Briefly, samples (1.0 g) were extracted with 0.01 N sodium hydroxide for 3 hrs in shaker and the supernatant was collected after centrifugation. To the known aliquots, 2 ml of trypsin solution was added and kept in a temperature bath at 37°C for 10 min. 5 ml of Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA) pre-warmed to 37°C was added. The reaction was terminated exactly 10 min later by adding 30% of acetic acid. A blank and control were also run simultaneously. The absorbance was read at 410 nm in UV-spectrophotometer (Shimadzu, UV-1800). Decrease of 0.019 of ∆A indicates the presence of 1µg of trypsin inhibitor in the sample.
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