I am using BAPA reagent to analyze the trypsin inhibitor activity in my legume flour. However, this method is doing problem. Any one who has a different method to analyze trypsin inhibitor activity or detailed method using BAPA reagent.
A cross check of the method is therefore needed, for selecting the critical step. Protein extraction, blanck reagent value and BAPNA source are potential problems. Refs in https://www.researchgate.net/profile/Mercedes_Pedrosa/publication/222622988_The_trypsin_inhibitors_present_in_seed_of_different_grain_legume_species_and_cultivar/links/0fcfd51025621b2bfc000000.pdf
Article The trypsin inhibitors present in seed of different grain
I haven't done this, but you could by Trypsin and a FRET substrate for trypsin and titrate the inhibitor by adding increasing amounts of the enzyme. If the trypsin inhibitor is tight binding, you can use the Morrison equation to calculate how much trypsin inhibitor you have.
Hi use tris-hcl ph 8, the given substrate, inhibitor and bovine trypsin provided by SIGMA. In time course monitor OD at 405 nm. Also do same without inhibitor as control.
@Muhammad Gulzar: hai brother even i am trying with BAPNA , its a very good method when compared to others its more specific to trypsin (even for papain), me to having few problems with bapna assay, it would be helpfull if someone helps me, i could understand its principle. after i did fplc(q sepharose ) for my protein i tested all fraction for trypsin activity. its showing inhibition in all fractions with few differences. i seriously could not understand where i go wrong, i would be thankfull to u if u share some ideas with us
Hi, I am currently doing this assay also on legume flour. Can I ask what kind of problems you experienced when you were doing it with your samples?
Hi, I am currently doing this assay also on legume flour. Can I ask what kind of problems you experienced when you were doing it with your samples?
Hi Dinesh kd, if you have any reference papers where the universal protease assay method is used to determine the protease inhibitory activity, could you please share with me? I actually want to use that method for some of my synthesized compounds to see their protease inhibitory activity.
Thanks in advance.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes