Protein Assays – Learn the Difference Between 280nm Absorbance, BCA, Bradford, Nano Orange and Lowry Assays

Because most people measure DNA—and care about protein interfering with that measurement–most people infer that protein absorbance can easily be measured at 280nm. This is not the case.

Eric Matthews Eric Matthews (3)

If you’re like most researchers, you get excited when you learn exactly how a new tool can be used to help you advance the boundaries of scientific knowledge. Some tools are so commonplace and used so frequently from our first days in the lab that they quickly become ho-hum. Before you realize it, you’d lost the chance to even wonder exactly why you’ve been doing the BCA assay instead of the Lowry assay for four years. On the other hand, perhaps that’s all you’ve wondered about. And after lab mates grew tired of you yelling “feel my heartbeat!” every time you learned why 750nm was more commonly used to measure Lowry than 800nm, it was suggested that you pursue a career involving the production and sale of microplate readers and microplate reader accessories.

The Bradford Assay is quick, simply and compatible with reducing agents—unlike the BCA and Lowry assay. However it is not compatible with high concentrations of detergents. The Bradford assay also does not accommodate high concentrations of protein, which might require users to dilute their samples before measurement.

The BCA assay is sensitive and works with both high and low concentrations of protein and isn’t bothered by detergents. It’s the perfect protein quantitation assay… if so many other compounds didn’t interfere with the BCA reagents. Phenol red, carbohydrates and other compounds affect performance, as do reducing agents commonly used in protein preparation.

The Lowry assay is effectively a simplified, BCA assay and it is slightly more sensitive than either the BCA or Bradford assays. The simplified chemistry makes the timing of the protocol a little more challenging to perform. Lowry assays have a list of interfering compounds that are slightly less problematic, if similar to, those of the BCA assay.

Because most people measure DNA—and care about protein interfering with that measurement–most people infer that protein absorbance can easily be measured at 280nm. This is not the case. Cystein-Cystein bonds, Tryptophan and Tyrosine absorb at 280nm–most other amino acids don’t. Since the concentration of those 280nm-absorbing-residues can vary by a factor of 400% or more from protein to protein, the calculated concentration of protein measured at 280nm can almost uselessly vary by just as much. Protein absorbance at 280 does have the benefit of being measured directly at low volume without dilution or other processing steps. But without foreknowledge of what you’re measuring at 280nm, it’s only useful if you aren’t very concerned with what the concentration of the protein actually is. To put a finer point on it, if you know you are measuring IgG or BSA, the concentration can be determined accurately and down to a very low concentration, but only if the protein you’re quantifying is characterized and reasonably pure.Fig. 1: Measure Protein Assays with the Microplate Reader FLUOstar Omega from BMG LABTECH

There are several fluorescent protein quantitation assays like NanoOrange or Quant-iT that eliminate many of the interference issues of other protein assays, though they remain susceptible to interference from a high concentrations of detergents. Because these kits use fluorescence detection instead of absorbance, the sensitivity of these kits can be orders of magnitude greater than the absorbance methods discussed above, but the expense of the kits may be higher, as is the complexity of the protocol. Fluorescent protein assays are most useful when the number of samples is high, miniaturization is possible or when the lowest limits of detection are important.

Nano Orange is a registered trademark of ThermoFisher Scientific

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