Protein Research Requires Protein Estimation;
But What is the Best Protein Assay?
Simple Answer: It depends!
There are a large number of protein estimation or quantitation assays available to researchers including UV adsorption at 280nm, modifications of Biuret Protein Assay, Lowry Protein Assay and the Bradford Protein Assay. This large number of assays makes it difficult for researchers to select the optimal assay for their protein solution. The choice of assay is normally dictated by the assay on the laboratory shelf!!
What Should You Consider Before Protein Estimation?
The best starting point to decide on the assay is your actual sample!
- What's in your sample solution, in addition to your protein?
- How concentrated or dilute is the sample?
- How much sample are you willing to sacrifice to an assay?
Secondly, look at the assay itself:
- Is time a consideration?
- Are you concerned by protein-to-protein variations?
- Do you have or need suitable protein standards?
- Is the right equipment available to you?
Running Interference!
Estimation of protein concentration usually occurs after the protein of interest has been removed from its native environment and is now in an aqueous solution. Not an easy feat in itself, ask any protein researcher about their run in with inclusion bodies! For a protein to be succesfully extracted into a solution requires numerous reagents that can have severe effects on subsequent protein assays. Reagents to be aware of are:
- Detergents: Crucial for hydrophobic or membrane proteins
- Metal Chelating Agents: Offer protection against enzymes
- Reducing Agents: To disrupt protein structure by reduce disulfide bridges.
- Salts: For stabilizing protein in solution
An example of the effects of an interfering agent on the assay is shown below. In this example, the assay was performed in the presence or absence of two commonly used detergents, the non-ionic Triton X-100 or anionic SDS detergents.
Detergents interfere with Bradford protein assays!
Researchers have two options. Review the Protein Assays Compatibility Chart, until you find a suitable assay that is compatible with all your solubilization agents, OR use an assay that cleans the sample before estimation!
Concentrate! My Precious!
A frustration and excitement of protein research is that the highly abundant proteins aren't really that interesting. The key players are often tightly regulated and expressed in low concentrations making the protein extraction and protein concentration very tricky. If this is the case, researcher's are often reluctant to use large volumes just to estimate protein assays.
The dilute nature of these low abundant proteins means a large volume is required for accurate estimation or the protein needs to be concentrated.
Researchers need assays that use small sample volumes, include a concentration step and are micro-plate formats.
The Protein Man Review
The major frustration of protein assays are crucial chemicals required in protein extraction and solubilization and the dilute nature of key proteins of interest. Assay compatibility charts offer one solution, but a unique solution is offered by G-Biosciences to remove interfering agents and concentrate proteins. Click here for a Handbook featuring our CB-X Protein Assay!
What are your biggest frustrations with protein estimation, please comment below, maybe Protein Man can help!
