Protein expression is the simple process through which proteins are changed, synthesized, and controlled in living organisms. The word may refer to either the object of analysis or the laboratory techniques used to produce proteins in protein research.
The experts who have worked with proteins are well aware of how vital the purity of protein and consistency are. They may even have to go through many issues that arise from a ‘poor’ protein test.
If the protein seems impure or is of inferior quality, it may lead to flawed specimens. It results in many problems that contribute to inconsistency and poor stability through tests—and may even trigger experiments to collapse entirely. If you are looking for ways to avoid such problems, keep reading to learn more about the parameters that may be used to test protein.
What are the perimeters for protein expression?
Many techniques involve measuring protein sample concentration to be accurate.
Though UV-Vis spectrophotometry, as well as Bradford assays, claim to be high-throughput ensuring it gets adopted in a majority of biochemistry labs, they are crude in comparison to enzymatic behavior assays. This crudeness happens because the effects of Bradford assays and UV-Vis are dependent on total protein in a study, not only the protein of concern. On the other hand, activity assays tend to focus on the target and provide the added advantage of calculating the level of active protein inside a distilled sample. Activity assay cannot quantify all proteins. You can opt for a reputed protein expression service provider to save your time by performing these tests for you.
2. Fluidic Analytics
MDS, or Microfluidic diffusional sizing, used in Fluidic Analytics’ Fluidity One method, is a quick and easy way to quantify protein concentration and size, to provide a good predictor of consistency. MDS employs microfluidic chips that guide a protein sample through a path where it can run alongside the auxiliary fluid inside a purely steady-state laminar flow, without any mixing. Proteins may only travel from one particular stream to another through diffusion, which happens at a rate equal to their size or hydrodynamic radius (Rh.) After some dispersal, both the streams are re-split, and then the proteins are marked. The ratio between diffused and undiffused light calculates the Rh.
MDS removes any of the drawbacks of the other technologies. Unlike electrophoresis, it removes contact between the protein and the matrix, and in their natural environment, samples can be run. The needed workflow is quick, with results in less than ten minutes using models with concentrations that can go as low as 10g/mL.
If the protein looks pure as per the technique described in previous points, it is time to verify its even distribution across the sample and not aggregating. DLS, or Dynamic light scattering, measures the degree of diffraction in any sample containing small molecules using polarized laser light. The hydrodynamic radius of particles present in the solution determines the amount of distribution that takes place as the selection passes through the device.
Though DLS is a simple technique that yields excellent qualitative results, it does not provide a complete image of size distribution within a protein sample because aggregates will quickly overpower a detector. The ease of use of DLS, coupled with its potential to expose the aggregate formation over a period of time, can make it a popular tool for determining homogeneity.
4. Size analysis
Mass spectrometry proves to be a very effective analytical tool that can detect post-translational changes with high sensitivity and specificity that are difficult to visualize using the above-listed methods. It operates by sorting proteins, also called peptides based on mass as well as charge, then accelerating them into a detector to provide a distinct range for each protein or even protein fragment.
One disadvantage of using mass spectrometry exclusively to determine protein content is the fact that it can be extremely low throughput along with needing thorough sample preparation. Furthermore, since the mechanism is denaturing and doesn’t detect misfolding activities, it is impossible to determine if proteins in the sample are intact.
5. Electrophoresis (Native/Denaturing PAGE)
Electrophoresis, like the quantification methods mentioned above, is commonly used by biochemists and can give a general image of both the size of your target protein and the presence of other protein-based impurities. However, you’ll want to get an idea of how concentrated the protein is before doing electrophoresis, lest you end up trying to rerun a gel on the weekend!
There are many electrophoresis methods, the most popular of which is denaturing SDS-PAGE. Since denatured with SDS (a detergent), samples are isolated by mass on a polyacrylamide gel matrix utilizing an electric field. Protein separation in native PAGE is more complicated and depends on the net charge, height, and form of the native framework. In all approaches, a smeared band indicates decay in a sample; however, this may also arise if the gel is saturated with protein.
The bottom line
Choosing methods to test the purity, size, and also overall consistency of any protein sample may be daunting; however, it can save you headaches in the long run. Although several techniques are now available, you can choose more than one at a time. All the methods listed above can be put together with another to offer the most precise and informative image of the protein samples’ condition.