Hello,
I'm looking to implement a fast method to screen anti-inflammatory products. Avoiding protein denaturation is linked to anti-inflammatory properties.
There is a nice number of papers describes a rapid and low cost screening method based on an incubation of the tests compound and a BSA solution (about 1 to 5% w:w) at higher temperature (usually 72°C) for a given time (between 5-20 min) buffered to ph6.4-6.6. Reading is done by spectrophotometry (usually 660nm). The method seems simple but when I try to implement, my reading is zero. I noticed that each laboratory introduces a slight change on the most cited protocol. I have introduced the major changes reported without any reading. Papers reporting the raw data indicates that after incubation the negative control (no test product of anti-inflammatory drug) DO is about 0.3-0.4.
Notice that all weightings, control and technicians have been investigated to try to find the root cause, unsuccessfully. Do anyone have any experience on this that can give me any advice?
Thanks
It sounds like this technique is intended to observe aggregation of BSA due to heat-induced denaturation by means of light scattering (turbidimetry), as measured by optical density. (How this phenomenon is related to anti-inflammatory properties of compounds is unclear, but never mind.) I think it would be important to start the experiment with a low baseline level of turbidity. I suggest you subject your BSA solution to ultracentrifugation before using it for the experiment in order to remove any preexisting aggregated protein.
Thanks, my problem is that i don't observe any aggregation. Theoretically it should be there after incubation.. but no!
It may be hard to see if there is a high background of aggregation. Reducing the background may make it easier to observe the effect.
Dear Adam,
Thanks for your comments but my DO reading is close to zero. No turbidity, no nothing a clear crystal solution.
Thanks again.
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