Denaturation assessments on egg albumin and bovine serum albumin suggest a link between tissue protein denaturation in living organisms and the onset of inflammatory complications. In this assay, a 5 ml reaction mixture containing egg albumin, phosphate buffer saline, and the test sample is heated, incubated, and cooled. Absorbance at 660 nm is recorded for both the reaction mixture and the standard (diclofenac sodium), with a phosphate buffer solution serving as the control.
Each time I conduct this assay, the absorbance readings consistently provide negative values. What could be the cause of this phenomenon? Why is the absorbance of the egg/bovine serum albumin denaturation assay taken at 660 nm?
This sounds like a measurement of turbidity (cloudiness). The long wavelength is used because the protein has no absorbance at that wavelength normally, but when it denatures and aggregates, the solution becomes turbid, which appears as an increased absorbance.
If you are instead seeing a decreased absorbance in the experiment, it suggests to me that the protein solution was already turbid before the heating step. After heating, either the solution clarified or the aggregated protein precipitated out of solution.
Take an absorbance spectrum of your protein before heating. It should peak at around 280 nm, and have no detectable absorbance at 660 nm. If this is untrue, I suggest you purify the protein by gel filtration chromatography or ultracentrifugation to remove aggregated protein.
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