Hello! I need to test anti inflammatory activity of a hydrogel by using protein denaturation assay.
We use BSA 5% in distilled water (pH 7.4). We prepared 3 samples (PBS 2.5 mL + BSA 450 uL, PBS 2.5 mL + BSA 450 uL + sample 50 uL, PBS 2.5 mL + BSA 450uL + aspirin 50 uL). Aspirin solution is 500ug/mL. pH of PBS 6.5. We denaturated samples using heat (60-80 °C for 15-30minutes).
We read at 278nm and 660 nm. The absorbance value is off, if we apply the formula the result is negative...
Could you give some citations that have a good protocol for this test? We do not know if the protein denaturates, the solution is clear even after 30min at 80°C for all samples. Would it be a good idea to centrifuge the solution after denaturation and before reading?
Hello, if no turbidity in the liquid after denaturation which usually happens you can't detect any changes by spectrophotometry. Maybe you need to increase the protein sample amount to make detectable any changes in turbidity during denaturation.
Use the standard protein like albumin or similar something and check the lower limit of detection of your method or system before applying a real sample.
Thank you for your suggestion! I did a uv vis scan between 200-700nm and our peak is at 278 nm but at 660nm, the wavelength used in literature, I had no detection..
I did serial dillutions for PBS+BSA sample and at 278nm and the results are ok. At 660nm the results are off, but in litrature they use 660nm to measure turbidity
BSA is an extremely stable protein with many disulfide bonds, and therefore may not be suitable.
You can try other common proteins such as hen egg lysozyme if protein identity is not important.
Alternatively, you can add detergents such as SDS to your assay, but they may change the initial absorbance.
Also, absorbance at 660nm pre-denaturation should be zero / near zero. An increase in abs. should be detected as a result of rising turbidity in the sample.
Good luck!
Thank you for your suggestion! I will try with hen egg since, yes, we did not managed to denaturate the BSA.
Hi, I tried too, sample groups:
1. BSA+PBS-unheated Abs: 0,521
2. BSA+PBS heated Abs: 0,41
3. BSA+PBS+Sample(Aspirin) heated Abs: 0,68
How do you think the results?
Hello!
I my opinion the results are good since the Abs of sample 3 is greater than sample 2.
But there is some difference between sample 3 and 1....in my opinion there should have been a smaller difference since you technically have the same amount of protein...
I am not an expert so mabe someone with more experience could bring more insight...
Good luck with your work!
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