Every protein quantification method reports a number relative to a standard, and most of the disagreement between laboratories comes from the standard rather than the chemistry. This page is about choosing the assay for the sample you actually have, knowing what interferes with it, and deciding what to calibrate against, so that two numbers taken a month apart mean the same thing.
The standard decides the number
A colourimetric assay compares your protein to a reference, and different proteins develop colour at different rates, so bovine serum albumin and immunoglobulin standards can differ by a wide margin on the same sample. If the protein of interest is available in pure form, calibrating against it removes the largest source of disagreement. If it is not, record which standard was used with every result, because a number without its standard cannot be compared with anything. An absorbance measurement at 280 nanometres avoids the standard entirely by using the extinction coefficient calculated from the sequence, which is why it is the right first method for a purified protein.
What interferes with which protein assay kit
Detergent breaks some assays and is tolerated by others; reducing agents interfere with copper-based chemistries; buffers with amines interfere with others; coloured or turbid samples break absorbance readings. The practical rule is to choose the assay from the buffer rather than from habit, and to run a spiked recovery in your own matrix before trusting a new combination. A dilution series that is not linear is the sample telling you the assay is being interfered with.
Absorbance, fluorescence and the useful range
Ultraviolet absorbance is fast, non-destructive and needs no standard, but it needs a clean sample and a known extinction coefficient and it reads nucleic acid as protein. Colourimetric assays work in dirtier samples at the cost of a standard curve. Fluorescent dye assays extend the range downward for small amounts and are the sensible choice when material is precious. Match the method to the concentration you expect rather than diluting a sample into the middle of an assay that was wrong for it.
Where the result is used, and what that demands
A number used to load a gel needs to be repeatable; a number on a certificate of analysis needs a method with a stated standard, a validated range and a recorded uncertainty. If the quantity feeds a dose or a specification, the method and the standard belong in the record with the result, and the reference materials that make such numbers traceable are published rather than invented in the laboratory.
Questions people ask about protein quantification
Which protein quantification assays should I compare?
Absorbance at 280 nanometres for a clean, purified protein with a known extinction coefficient; a colourimetric assay chosen for your buffer when the sample is dirty; a fluorescent dye assay when the amount is small. Choose from the buffer and the expected concentration, not from habit.
Why do two laboratories report different concentrations?
Usually the standard rather than the method. Different reference proteins develop colour differently, so the same sample can read high or low depending on what it was compared against. Record the standard with every result.
Does absorbance need a standard curve?
No, which is its advantage: it uses the extinction coefficient from the sequence. It does need a clean sample, because nucleic acid and scattering both read as protein.