Measuring what a protein preparation actually contains: why a protein analyzer is a category rather than an instrument and why protein purity analysis needs an orthogonal pair rather than a single method, what size exclusion separation reports about aggregates that a gel cannot, where protein sequencing by mass spectrometry confirms identity that a blot only suggests, what gst tagged protein purification and other tag routes leave behind that has to be measured, how a hydrophilic pvdf filter and similar filtration choices lose protein you did not know you had, and what a certificate of analysis should state before a preparation is used in anything quantitative

Characterising a preparation

1. Use two orthogonal purity methods. A denaturing gel and a size exclusion separation see different problems: the gel finds truncations and contaminants by size, the column finds aggregates and multimers. Either alone gives an optimistic answer.
2. Confirm identity independently. A band at the right size is consistent with the protein and does not identify it. Mass spectrometry, either intact mass or peptide mapping, is what confirms it, and it is worth doing once per construct.
3. Measure concentration by a stated method. Absorbance with a calculated extinction coefficient, a colorimetric assay against a standard, or amino acid analysis give different numbers. Choose one, state it, and use it consistently.
4. Measure the residues that matter downstream. Imidazole, reducing agents, detergents, endotoxin and residual protease each interfere with something. Measure the ones your assay cannot tolerate rather than all of them.
5. Account for filtration losses. Protein adsorbs to membranes, and a sterile filtration step can remove a meaningful fraction of a dilute preparation. Low binding membranes exist and the loss should be measured once.

Aggregates are the invisible impurity

Aggregated protein runs as monomer on a denaturing gel and behaves very differently in every biological assay. It is the impurity most likely to be present and least likely to be measured.

A size exclusion run on each preparation takes fifteen minutes and is the single most informative addition to a characterisation panel.

A certificate should say what was measured

Concentration by a named method, purity by two methods, aggregate content, identity confirmation, endotoxin where relevant and the final buffer. Those six lines make a preparation reusable and comparable.

Insist on them from suppliers and produce them internally. It is what turns a preparation into a reagent.

Common questions

Is a single band on a gel enough?

No. A denaturing gel is blind to aggregates and to species of identical size, and it is a low resolution method. Pairing it with a size exclusion separation catches most of what it misses.

How should protein concentration be measured?

By a method suited to the protein and stated with the result. Absorbance is accurate for proteins with aromatic residues; colorimetric assays are matrix and standard dependent; amino acid analysis is the reference.

Why does my dilute protein disappear on filtration?

Adsorption to the membrane. It is substantial for dilute solutions, and low binding membranes plus a measured recovery are the practical response.

Browse by service class

• Compare Gene synthesis on advertised prices