12/7/2017 How to Measure Protein Concentration More Accurately - Bitesize Bio

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How to Measure Protein Concentration More Accurately
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By Jennifer Cable (https://bitesizebio.com/pro le/jennifer-cable/)
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When working with proteins, one key part of any good assay is accurately determining how much protein you have. Accurate
quanti cation is obvious important if, for example, youre trying to determine a binding constant or measure enzyme kinetics; but even
if youre doing something more qualitative, having a good idea of how much protein you have will enable you to compare results from
one experiment to the next and from one protein to the next.

There are several ways to measure protein concentration, and each of them has its own advantages and disadvantages, so how do you
know which method is right for your protein? In this article, were going to discuss three di erent methods for measuring protein
concentration: absorbance at 280 nm, the Bradford assay, and the BCA assay.

Absorbance at 280 nm (https://bitesizebio.com)

How it works: Aromatic residues, like tyrosine and tryptophan, absorb UV light at 280 nm. So, if you have an extinction coe cent for
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your protein (e), you can measure the absorbance in a UV/Vis spectrometer and calculate the concentration of your protein using
Beers law (A = elc, where l is the path length of the spectrometer). You can estimate the extinction coe cient of your protein based on
the sequence using Expasys ProtParam tool (http://web.expasy.org/protparam/). Because ProtParam only considers the linear
sequence of your protein and doesnt take into account the structure, which can a ect the extinction coe cient, youll want to
denature your protein before you measure the absorbance. (I like to denature proteins in 6 M guanidinium.)

Advantages: This technique is quick and doesnt require any special reagents, except for the guanidinium, which you may have on
hand anyway.

Disadvantages: This method relies on having an accurate extinction coe cient for your protein, which depends on the number of
aromatic residues. If there arent a decent number of aromatic residues, your extinction coe cient will be quite low, and you will need
a fairly concentrated sample to get a reasonable absorbance (generally an absorbance between 0.1 and 1.0 is considered within the
linear range). Also, ProtParam warns that there may be at least a 10% error in the extinction coe cient if there are no tryptophans in
your protein. Therefore, if your extinction coe cient is low, which is likely the case if there are no tryptophans in the sequence, a 10%
error could signi cantly throw o your assessment of the nal protein concentration.

Bradford Assay
How it works: The Bradford assay is a colorimetric assay based on the interaction between Coomassie brilliant blue (you know, the
stu you stain your gels with) and the arginine and aromatic residues in your protein. When the dye binds to these residues, its
maximum absorption shifts from 470 nm to 595 nm. In general, you measure the absorbance of a series of known concentrations of a
standard protein, generally BSA, and create a standard curve. You then use that standard curve to calculate the concentration of your
protein sample based on its absorbance.

Advantages: This assay is quick, and the reagent is not a ected by the presence of reducing agents, like DTT and ?-mercaptoethanol,
that may be in your bu er.

Disadvantages: Basic conditions and detergents, such as SDS, can interfere with the dyes ability to bind to the protein; however,
there are detergent-compatible Bradford reagents.

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12/7/2017 How to Measure Protein Concentration More Accurately - Bitesize Bio

Also, like the absorbance at 280 nm technique, the Bradford assay depends on the sequence of your protein. If your protein doesnt
contain a decent number of arginine and/or aromatic residues, then the dye will not bind to the protein as e ciently, resulting in an
underestimation of your protein concentration.

Finally, this technique depends on comparing the absorbance of your protein to that of a standard protein. So, if your protein doesnt
react to the dye in a similar manner as your standard protein, your concentration can be o .

BCA Assay
jHow it works: The BCA assay is another colorimetric assay like the Bradford assay. It makes use of the biuret reaction, in which the
protein
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14 backbone chelates Cu2+ ions and reduces them to Cu1+ ions. The Cu1+ ions then react with bicinchoninic acid (BCA) to form a

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purple-colored product that absorbs at 562 nm. The procedure is similar to that of the Bradford assay, in which you create a standard
curve based on a series of known protein standards.
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Advantages: Because the peptide backbone is involved in the reaction, the BCA assay is less sensitive to the types of amino acids in

fthe protein. However, the reaction is in uenced by cysteine, tyrosine, and tryptophan residues.
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Share reagent is not sensitive to detergents and denaturants, so its okay to have those in your bu er.

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Disadvantages: The presence of reducing agents in your bu er can interfere with the dye, but there are reducing agent-compatible
dyes available.
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The reaction takes some time to proceed. Usually, the samples are incubated at 37C for 15-30 min. Also, as in the Bradford assay,
you determine your protein concentration by creating a standard curve from a known, standard protein. So again, if your protein
doesnt interact with the dye in a similar way as the standard protein, your concentration could be o .

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So there you have it: a few things to consider when choosing a technique to measure protein concentration. Choosing the right
technique for your protein will take a bit of trial and error, but having a good technique in your back pocket to accurately measure your
protein concentration will save you a lot of time and energy and help you get more reproducible results.

Whats your favorite way to measure protein concentration?

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3 Comments

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12/7/2017 How to Measure Protein Concentration More Accurately - Bitesize Bio

Barrack on November 23, 2017 at 7:10 pm

How can I cite this article?

Reply (https://bitesizebio.com/10178/how-to-measure-protein-concentration-more-accurately/?replytocom=114052#respond)

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Riya on July 29, 2017 at 7:54 pm

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Hello,
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I want to measure blood protein concentration, which method would be best for this?
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Reply (https://bitesizebio.com/10178/how-to-measure-protein-concentration-more-accurately/?replytocom=106899#respond)

Ana on March 18, 2017 at 10:25 pm

hello,

For my protein sample, I have an extinction co-e cient of 1.5308

would you describe this as low?

thank-you in advance

Reply (https://bitesizebio.com/10178/how-to-measure-protein-concentration-more-accurately/?replytocom=97753#respond)

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