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 recognizes 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.
A protein analysis service, and what protein analysis services cover
Protein analysis services cover a ladder of questions and it pays to ask for the answer rather than the technique. Identity and sequence come from mass spectrometry, intact mass and peptide mapping. Purity and aggregation come from size exclusion, capillary electrophoresis and light scattering. Higher order structure comes from circular dichroism, differential scanning calorimetry and hydrogen deuterium exchange. Post-translational modifications need their own mapping, glycans released and profiled separately. Quantification and activity need an assay matched to the molecule. A quote is comparable only when the deliverable, the report and the method are named.
intact mass, and what it settles
An intact mass measurement weighs the whole protein rather than its peptides, which confirms the sequence's molecular weight, shows the glycoform distribution and catches an unexpected modification or clip that a peptide map can average away. Deconvolution of the charge envelope is where the error usually enters. It is the first analytical question asked of a new batch.
lc ms amino acid analysis for composition and content
lc ms amino acid analysis hydrolyses the protein and measures the released residues, which gives an absolute protein content independent of a dye binding assay and is the anchor other assays are calibrated against. Hydrolysis destroys some residues, so the method states what it cannot report. It is also how a peptide's counter-ion and water content are resolved.
antibody concentration, and the three ways it is measured
An antibody concentration is read by absorbance with a sequence derived extinction coefficient, by a dye binding assay against a standard, or by amino acid analysis, and the three disagree by several percent on the same sample. For a reagent the absorbance route is usual; for a release specification the method is fixed and stated. Aggregate content changes the absorbance reading without changing the mass.
drug antibody ratio and how it is determined
The drug antibody ratio of a conjugate is measured by absorbance where the payload has its own chromophore, by intact mass, or by a chromatographic separation of the species, and the distribution rather than the average is what matters for activity and safety. Two conjugates with the same average can differ entirely in their unconjugated and highly loaded fractions.
antibody drug conjugate analysis as a panel
antibody drug conjugate analysis is a set of measurements rather than one: the loading distribution, free payload, aggregation, charge variants, binding and potency. Each has a method with its own bias, so a supplier comparison is only meaningful method by method. Free payload is the measurement with the most direct safety consequence and the tightest limit.
antibody isotyping and why it is asked
antibody isotyping identifies the class and subclass of an antibody, which decides the secondary reagent, the purification chemistry and the effector functions it will have. For a hybridoma it is a quick kit; for a sequence it is read directly. A surprise result usually means a mixed population or a mislabelled vial rather than an unusual immune response.
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.