I've always conducted biuret assay by using a spectrophotometer. Recently, I have tried using a micropate reader for it. It is faster and requires less reagent and BSA. But it seems that the results are a bit higher with it than with spectrophotometer. The proteins I examine with biuret assay are about 190_260 mg/ml that I dilute 40 times before the reagent addition.
I would not care much about absolute values (check your dilutions, and there might be variations between machines due to filter bandwidth and calibration), as long as they are plausible and within a valid range. However, while cuvettes have a well-defined thickness of the liquid layer (usually 1cm), in microplates, the layer thickness depends on the volume that you have in a specific well. So you are introducing an additional source of error by variations in the volume by pipetting. If required, I'd recommend using another suitable wavelength as reference for reducing/eliminating this error.
If you have 200ul in a micro plate, the depth of liquid will be 0.6-0.7 cm. Your absolute values will therefore be less in a microplate than a cuvette. I assume you are referring to extrapolated values for protein conc when you say that they are higher than with the cuvette. Micro plates are very convenient and probably most people use them these days, but you cannot expect the data to be quite as accurate, as noted by Wolfgang Schechinger above.
Nick is correct but you are reporting a systematic bias as well (presumably) as increased scatter
@Nick Gee
Thanks for your response. Yes the values are less but the protein concentration is higher.
So as I understood, it is better to use the cuvette to get more precise results. Isn't there a way to decrease the mentioned error in microplates dear @Paul Miham?
Dear @Wolfgang Schechinger, thanks for your response.
how can I find the best wavelenght?
- Badges
- Science method