Have you ever spent ages correctly quantifying your protein samples, cast the perfect gel, efficiently transferred, probed and eventually come to the visualisation stage only to be presented with a loading control like this:
Well before you throwing your blot out with the bathwater and repeating the experiment you should maybe consider your choice of protein assay and how this can interact with your samples.
Choosing a protein assay
Most modern protein assays are based on two colorimetric assays. The first which relies on the interaction of proteins with copper and the subsequent reduction of these copper ions (Biuret and its derivations BCA and Lowry). The second on direct alterations to Coomassie Blue dye due to its interaction with proteins in solution (Bradford).
The biuret test was first described in 1833, although its usage as a protein assay came much later. At its most simple an aqueous protein sample is treated with as strong base (typically sodium hydroxide NaOH) and a copper(II) sulfate solution. When proteins are present the copper(II) binds with nitrogens present in the protein sample. These liberated copper(II) molecules can now be reduced which in turn changes the color of the solution from a pale blue into a nice shade of purple as seen below. As the amount of reduced copper ions are correlated with the amount of protein present in the sample, and in conjunction with a standard curve it is then possible to accurately quantify proteins within the range of 5-150 mg/mL.
Given its relative insensitivity (how many of us are using samples in the range of 5-150 mg/mL) and lack of reactivity with single amino acids or small peptides the biuret assay is ideal for high protein samples such as tissue.
A positive biuret assay sample. Image c/o Philip Evans
There are two derivations of the biuret assay which be used to dramatically increase assay sensitivity.
Bicinchoninic acid (BCA) Assay
In the BCA assay the copper ions generated with through interaction with proteins react with BCA again producing a strong violet colour. The difference however is that the formed BCA/copper complex absorb light much more strongly than those generated by the biuret assay alone greatly increasing the assays sensitivity. With proper sample preparation and accurate standard curves it is possible to assay samples accurately within the 0.001 to 2 mg/mL protein concentration range.
The Lowry assay is an earlier derivation developed by Oliver Lowry in the 1940’s. In a similar fashion to the BCA assay the Lowry assay relies on the reaction of copper ions, produced from the interaction of copper(II) sulfate with proteins present in the sample, with a further reagent. In this case Folin–Ciocalteu reagent with the sample becoming blue in colour when higher levels of protein are present.
From left to right, decreasing protein concentrations as assessed by the Lowry protein assay. Image c/o Imgur
However, all three assays described above are sensitive to reducing agents such as dithiothreitol (DTT) or beta-mercaptoethanol (BME) which are often used in sample preparations. These reducing agents can inflate protein concentration values by increasing the reduction of copper ions.
Enter protein-dye assays which are not sensitive to reducing agents, although are sensitive to detergents such as Triton, Tween or sodium dodecyl sulfate (SDS).
The Bradford assay relies on an absorption shift by Coomassie Blue when it binds to proteins present in a solution. Under acidic conditions unbound Coomassie will present its typical blue-green color with a maximal absorbance of 465 nm. However under the same conditions Coomassie will readily binds non-covalently with the carboxyl groups of proteins which induces a conformational shift allowing for a further ionic bond to form between the protein and the Coomassie dye. This binding alters the absorbance profile of the dye, which can be measured at 595 nm. By measuring the shift from 465 nm to 595 nm and in conjunction with an appropriate standard curve accurate protein quantification is possible.
Which assay to chose?
So if you see odd looking results even though you’re sure your quantification is accurate and your loading control is correct, consider looking at your protein assay. If your extraction buffers contain reducing agents such as DTT or BME consider the Bradford protein assay. Conversely if you samples contain detergents then the BCA or Lowry assay may be preferred. Finally if working with high concentration samples the biuret assay may be used, but it is often preferable to simply dilute high concentration samples down.