Hi everyone,
I'm using the Lanvaya et al. (2010) protocol for testing in vitro anti-inflammatory activity. I add 450 μl of a 0.5% BSA solution to 50 μl of test samples. The mixture is then incubated at 37°C for 20 minutes, followed by a temperature increase to 57°C for 3 minutes. After cooling, I add 2.5 ml of phosphate buffer. However, I have a few issues:
Which blank should I choose? distilled water or Phosphate buffer or mdthanol since my test simples are prepared in methanol.
And what is the appropriate length wave? I read absorbance with 255 nm , some protocols use 416 nm, or 660. My tubes are little bit turbide.
Thanks for your answers.
Cordially. Ouiza
I think what you really need for this assay (thermal stability of BSA in the presence of a compound) are 2 controls, rather than a blank. The two controls are (1) no compound, just the solvent that the compound is dissolved in, and (2) a standard compound that has the maximum effect in the direction your compound is supposed to work, dissolved in the same solvent. You can then compare the turbidity to the turbidity of the controls, with the controls defining the minimum and maximum points of the effect. The treatment of the controls should otherwise be exactly the same as the treatment of the experimental samples in every respect.
Turbidity increases sharply as the wavelength decreases, which means the assay would be more sensitive when using the shorter wavelength. However, organic compounds usually have an absorbance in the UV, which causes interference with the measurement, so it makes sense to use a longer wavelength at which the compound's absorbance does not interfere. For colorless compounds, any wavelength in the visible range (about 400-700 nm) would be adequate. 416 nm would be more sensitive than 600 nm for detecting turbidity, but 600 nm would be less likely to suffer interference from compound absorbance. It's a tradeoff.
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