Poor quality protein samples can result in low quality data and wasted time in downstream applications. Protein quality is influenced by a number of factors, including age, storage conditions (including temperature, buffer formulation, number of freeze thaw cycles etc.), and the method of purification. Further to this, it is rarely possible to fully ascertain the nature of a protein using just a single technique - and researchers can end up working with incorrect assumptions about their protein.
For many biophysical/structural characterization experiments, correct interpretation relies on the assumption that:
- The measured concentration of the protein is accurate
- Protein samples are pure and homogeneous
- All of the protein is solubilized and in a natively active state
In the 2014 paper “Quality assessment and optimization of purified protein samples: why and how?”, Raynal et al propose a series of steps to determine the quality of a purified protein sample, through which researchers can assess the purity, integrity, homogeneity, and activity of their protein.
The Fluidity One offers an additional approach to the characterisation techniques recommended in the paper and, due to it’s speed, minimal sample requirements, and ability to quantify proteins without aromatic residues it may in some cases offer a more appropriate alternative, particularly for time sensitive applications.
Understanding of total protein concentration in a sample can be used to deduce the percentage of active protein (if active protein concentration has been determined), which is important for the interpretation of experiments. Commonly used protein quantification methods include UV-Vis spectroscopy or comparison to calibration standards in Bradford or Lowry assays.
In certain situations, the Fluidity One offers a number of advantages over UV-Vis spectroscopy and colorimetric assays for protein quantification:
- Proteins or peptides with no aromatic residues can be detected - Fluidity One uses an amine reactive, fluorogenic dye that will react with the N-terminal amine and the lysine residues of a protein.
- Low concentration requirements - the Fluidity One can quantify proteins down to 10 ng/µL.
- Low sample volume requirements - samples can be quantified with a high level of accuracy using just 5 µL, preserving precious samples.
Provided the anticipated hydrodynamic radius of a full length, correctly folded protein is known, measurement of hydrodynamic radius on the Fluidity One can provide an indication of sample monodispersity. Critically this assessment can be made in 6 minutes with just 5 µL of sample - in contrast to SEC-MALS which can take ~30 minutes and >5 µL sample, often at high concentrations.
A lower than expected hydrodynamic radius can reflect degradation or truncation of the purified protein whereas a higher than expected hydrodynamic radius may be a consequence of oligomerisation or aggregation.
As with Dynamic Light Scattering (DLS), the Fluidity One does not resolve multiple species in a single protein preparation. However, unlike DLS, the Fluidity One gives an average hydrodynamic radius of all proteins in solution.
DLS is an excellent tool for detecting even trace amounts of aggregates in a sample as a 60 nm radius particle scatters 1 million times more light than a 3 nm one. However, as even a few percent of larger aggregates in a sample can swamp the signal coming from small particles, this technique is ill-suited to characterising samples where particulate matter is common. In these instances, the Fluidity One allows researchers to understand the true heterogeneity of their protein prep.
To ensure experimental reproducibility, lot-to-lot consistency of protein preps should be routinely assessed. Raynal et al point out, however, that once the quality of a purified protein sample has been fully assessed and optimized, repetition of the whole quality control workflow is not necessarily required for each subsequent use.
In this context, the Fluidity One measurement of hydrodynamic radius and concentration can provide a rapid assessment of the consistency of a batch prior to commencing more time consuming or costly analysis.