15 Methods for Protein Standard Curves and SDS-PAGE
The method for generating a standard curve and measuring concentrations of samples is clearly outlined in the Bradford Assay Manual. Anticipate a working range of 100-1,500 µg/mL protein. Standard bovine serum albumin samples will be provided.
Caution: The Bradford Coomassie solution is a very strong acid (phosphoric acid)—be very cautious when working with it, and if exposed wash immediately with water. Collect used reagent in appropriate waste container in the fume hood.
When preparing and running the SDS-PAGE gel of the purified protein sample, the only difference lies in sample preparation. Approximately 5-20 µL of desalted protein sample can be mixed with 20 µL of 2x SDS, which can then be heated at 100 °C for 5-10 minutes. Briefly centrifuge the tubes to get the entire sample to the bottom of the tube. 20 µL of this mixture can be loaded into the wells of the gel. A rule of thumb for mini gels is to load about 0.5 microgram protein per expected band, so the gel is not overloaded.
Deep Thoughts on Protein Concentration
There are many ways you can measure protein concentration and, as is often true, each way has its advantages and disadvantages. How are you measuring protein concentration? The reagents for a Bradford Assay are provided, but you should still consider whether this is the best method for measuring the concentration of your protein. In addition to the Bradford Assay, two of the most common ways to determine protein concentration are the BCA Protein Assay and Absorbance at 280 nm using a Nanodrop or Picodrop.
A few things to consider when selecting a method include compatibility with the sample type and buffer components (including added zinc), assay range and required sample volume, speed and convenience for the number of samples to be tested, availability of spectrophotometer or plate reader to measure the absorbance by the assay, utility in measuring whole cell lysate or pure protein, uniformity (how much protein-to-protein variation there is) and, if used, appropriateness of the standard. In your case, you’ll also need to consider the effect adding a non-canonical amino acid to your protein might have on your measurements.
The Bradford Assay relies on the proportional binding of Coomassie dye to proteins. Within the linear range of the assay, the more protein present, the more Coomassie binds, and the darker the color of the sample becomes. Coomassie absorbs at 595 nm so using a protein standard, most commonly BSA (bovin serum albumin), you can generate a standard curve and use this to measure the protein concentrations of your samples.
The BCA Protein Assay is also a colorimetric assay used to quantify protein concentration. This method relies on the reduction of copper and subsequent detection of the reduced copper by BCA (bicinchoninic acid). This BCA/copper complex exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. Like with a Bradford assay, a protein standard, most commonly BSA, is used to generate a standard curve with which to determine the protein concentrations of your samples.
A Nanodrop is a common lab spectrophotometer that can be used to determine protein, DNA, and RNA concentration. To measure protein concentration, the Nanodrop uses absorbance at 280 nm, often referred to as A280, and an extinction coefficient to determine concentration of a pure protein sample. What’s an extinction coefficient? An extinction coefficient is a constant for a protein reflecting how much UV light it will absorb based on its amino acid composition; aromatic amino acids are primarily responsible for UV-light absorption. The Nanodrop has the advantages that it requires only very small sample sizes (~1-2 µL) and is fast.