Amylase

By A2-33  [CC BY-SA 3.0], via Wikimedia Commons

We recently published a new application note on protein characterization using our latest SEC system, OMNISEC, and I wanted to bring your attention to this elegant piece of work that beautifully shows the kind of information you can get from a multi-detector system and, perhaps more importantly, the kind of information you might be missing from a single-detector, UV-based system.The chromatogram below shows a typical chromatogram of a standard protein,    β-amylase:

chromatogram of a standard protein, β-amylase

It looks pretty typical with a monomer and a small amount of aggregates, right? Wrong!

Let’s looks again with a multi-detector system and the calculated results in the table below:

multi-detector chromatogram of a standard protein, β-amylase

multi-detector protein analysis

Suddenly we have a great deal more information and the picture changes dramatically.

The information from the molecular weight detector (light scattering) tells us that this isn’t actually a protein aggregate at all but a protein of similar molecular weight.  Measuring its size using the molecular weight and IV detectors tells us that the first peak is much larger despite its similar molecular weight and it’s this difference in structure that explains the different retention volumes, despite their similar molecular weight.

So is it a structural variant of β-amylase, or a different protein entirely?  Well if it was a structural variant, it would have a similar, UV absorbance for its concentration but these two peaks have significantly different levels of absorbance compared to their concentration (measured by the RI).  This tells us that it is a contaminating protein, rather than a structural variant.

So what looked like a simple protein with some aggregates is actually far more complex.  This sample contains the monomer and a second contaminating protein of similar molecular weight but a much less globular structure.  This protein accounts for almost 30% of the sample.

…and that was just a standard protein.  Just think about what you could learn about your own protein with the power of multi-detection.

If you are interested in reading further, please click here to download the full application note.

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