I'm performing the Bradford protein assay using a spectrophotometer with four cuvette slots. I have six samples to analyze. Here's how I proceeded:
Slot 1: Reagent blank (Bradford reagent + water, no protein)
Slots 2–4: Samples 1, 2, and 3
After reading the first three samples, I removed them and placed samples 4, 5, and 6 in the same slots.
I kept the blank cuvette in Slot 1 throughout.
However, by the time I inserted the second set of samples, the absorbance reading of the blank had increased to 0.18.
My question is: Should I press "blank" again to re-zero the instrument, or is it okay to blank only once at the beginning and continue measuring all samples using that reference?
Any insights on best practices for handling this kind of situation would be greatly appreciated!
Dear Haseeb, since absorption of test samples are always taken in reference to blank,so every set of reading should be started from setting blank as zero. This prevents you from minute differences per set of reading taken.
You should always press blank in all of your measurements.
You should re‐blank before measuring your second batch. The Bradford reagent may drifts over time (and with temperature), so whenever your “zero” drifts by more than ~0.01 AU, pop the blank back in, hit “blank,” and then run your next samples.
I agree with the other comments that you should re-blank, since the Bradford assay has no set endpoint, so the color continues to develop while you're measuring your samples. Also note that the blank should not be made with water, it should be made with whatever buffer you dissolved/diluted your protein samples in.
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