Protein characterization is a set of separate questions that are often ordered as one package: is this the right protein, how much of it is there, what else is in the tube, is it the size and state you expect, and does it still work. Each question has its own methods, its own failure modes and its own price, and a characterization package that answers four of the five can look complete on an invoice. This page sets out what proves what, so a request can be specified rather than delegated.
Identity, and why one method is rarely enough
Identity is established by sequence evidence and confirmed by mass. Peptide mapping with mass spectrometry gives coverage of the sequence and finds modifications; intact mass confirms the molecule as a whole and catches truncation and unexpected glycosylation; amino acid analysis and terminal sequencing answer narrower questions. An immunoassay confirms that something the antibody recognises is present, which is not the same as identity. A request that asks only for a gel and a Western is asking for a presence test.
Purity is a question about the impurity you care about
There is no single purity number. Size exclusion chromatography reports aggregate and fragment; reversed phase and ion exchange report charge and hydrophobic variants; a host cell protein assay reports what the cells left; an endotoxin test reports pyrogen load; residual DNA and leached ligand are their own assays. Each is a different denominator, so a specification has to name which impurities matter for the intended use rather than ask for a percentage.
Size, state and higher order structure
Nominal molecular weight is the easy part. What usually matters is whether the protein is monomeric in the buffer you will use it in, whether it aggregates on freezing, and whether it is folded. Size exclusion with light scattering gives absolute mass and aggregation state, analytical ultracentrifugation resolves what chromatography cannot, and spectroscopic methods report secondary structure and thermal stability. These are the methods that explain a batch that passed purity and failed in the assay.
Activity is the only method that answers the real question
A protein can be the right sequence, the right mass, monomeric and pure, and inactive. A potency or binding assay is the one measurement that speaks to what the protein is for, and it is also the one most often left until the material has already been accepted. Specify it first and let the physical methods explain its result, rather than the other way round. For material heading into a regulated study the specification framework expects both sets, and the physical methods exist to make the activity result interpretable.
Questions people ask about protein characterization
What should a characterization package include?
Identity by sequence and mass, the purity assays for the impurities that matter to your use, size and aggregation state in your own buffer, and an activity or binding measurement. Ask which of those is missing rather than accepting a package name.
Is a gel and a Western enough?
They show that something of about the right size is present and that an antibody binds it. That is a presence test, not identity or purity, and it will not detect variants, aggregate or loss of activity.
Why did a batch pass purity and fail in my assay?
Most often aggregation or misfolding, which purity by area percent does not report, or loss of activity that no physical method measures. Size with light scattering and a potency assay are what separate those two.