Methods are based on Chel-Guerrero and colleagues'Article Phytochemical Profile, Toxicity, and Pharmacological Potenti...
.We use a 1M Tris buffer with pH of 6.8, with Bovine Serum Albumin as the protein for the assay. Our control sample is at a 500μg/mL BSA concentration. We incubate 1mL of our samples in a dry bath at 72°C for 10 minutes, then let it rest for another 10 before testing its absorbance at 660nm.
The issue is that even after following the provided method, there is no absorbance reading at 660nm. We believe this is likely because no protein denaturation has occurred. We have tried other methods of heating our samples, like with a water bath and in an oven, to no success. We have tried heating the sample for another 10 minutes after letting it rest, thinking it would have the same effect as heating it for 20 minutes straight (we let it rest to put it through the spectrophotometer). We have yet to try heating it for 20 or even 30 minutes straight. We would also like to try heating it first for 37°C for a time, then heating it to 72°C.
Is it possible for BSA to not denature at all when exposed to 72°C heat for 10 minutes?
Any advice would be greatly appreciated.