51Why does the Bradford assay respond differently to different proteins?52What is the basic principle of the Lowry assay?53Why do colorimetric protein assays require a standard curve?54What might cause a flattened spectral peak?55Why can absorbance be negative?56What should I check for a jagged spectrum or large differences between repeats?57Why can different instruments give different results for the same sample?58Is OD600 truly absorption?59How can OD600 be converted to cell count?

Why can absorbance be negative?

Negative absorbance means the sample measurement transmitted more light than the reference measurement; it does not indicate a substance with negative concentration. Possible causes include a more absorbing blank, matrix mismatch, residue or reference drift.

Save the spectrum and check buffer matching. Clean the measurement surfaces according to the instrument instructions, establish a fresh matched reference and remeasure blank and sample.

Small fluctuations near zero should be assessed against blank noise. Persistent negative regions require investigation before quantification. Clipping values to zero does not resolve the cause.