With so many protein assays to choose from, each with its own advantages and limitations, you have to consider their suitability for the application. In some instances, it can be necessary to utilize more than one type of assay. Here, we discuss how to select the most appropriate protein assay.
There are 3 main types:
Dye Binding Assays
These bind protein molecules to Coomassie dye under acidic conditions.
- Dye based protein assays show the largest protein-to-protein variation and in some cases, these assays show no protein response as no protein-dye complex is formed.
- Protein solutions containing surfactants (detergents) interfere with the dye-based protein assays.
- Most dye-based protein assays require preparation of standard plots.
- Since most colorimetric protein assays require at least 0.5µg proteins for a reliable estimation, dilute protein solutions require a larger volume to reach the limit of detection for the protein assay.
Copper Ion-Based Assays
These bind cupric ions with peptide bonds resulting in their reduction to cuprous ions.
- Assays involving the reduction of cuprous ions to cupric ions have significant protein-to-protein variation.
- Protein solutions containing reducing agents, metal chelating agents, dyes, amines, and sugars, cannot be estimated with the protein assays based on copper ions.
- Most copper ion-based assays require preparation of standard plots.
Test Strip-Based Assays
A chromatographic capture method that estimates the amount of protein in the sample.
- The test strip based dotMETRIC™ protein assay, based on the chromatographic capture of the proteins, is independent of the primary structure of the protein and hence shows little or no protein-to-protein variation.
Within these different types of protein assay methods, there are a number of specific assays to choose from – for example, the Lowry assay method, Bicinchoninic Acid (BCA) method, or the Bradford assay.
Protein Assay Selection and the Nature of the Protein Sample
The nature of the protein sample and its compatibility with the protein assay method is of utmost importance during the assay selection process. The success of your method revolves around several factors you must take into consideration in order to select the method that is most compatible with the samples being analyzed. Factors to consider in selecting an appropriate protein assay include:
- interfering agents
- sample preparation, size, and dilution
- assay sensitivity
- time considerations
- protein standards and protein-to-protein variations
- instrumentation requirements
- understanding and controlling the particular assumptions and limitations that remain
It may be useful to ask these questions when selecting a protein assay method:
- Is the sample hard to obtain?
- What size of the protein sample is required?
- How much time do you have?
- Can your sample be easily solubilized in a compatible buffer?
- Do you have a way of minimizing interfering agents?
- What protein standard will you use?
- Are you taking protein-to-protein variations into consideration?
- What kind of instrument requirements are there?
Each Method has its Own Specific Advantages and Disadvantages
With all the different assays out there, it’s important to understand the basics! The goal is to select a method that requires the least amount of manipulation or pre-treatment of the samples to accommodate substances that interfere with the assay. Because each method has its own specific advantages and disadvantages, no one reagent is considered the “best” protein assay method. Since one size does not fit all in the lab, most researchers have more than one type available.
G-Biosciences Has All Your Protein Assay Supplies
At G-Biosciences, we have protein assay supplies to meet a wide array of applications. Browse through our products and then contact us with any questions or to place an order.