31 Proteomics, Genomics & Microarrys
Figure 2. Correlation functions obtained at different θ values. Second decay becomes obvious at low-angles.
Figure 4. DLS of chemically stressed monoclonal antibody produces a correlation function with between 2-3 major components (dominant component is aggregated protein). Free monomeric antibody is still fully resolvable from aggregates. The average hydrodynamic diameter is, of course, infl ated in this case (dh eff >> 12 nm).
Figure 3. Dilute monoclonal antibody measured at a ninety-degree scattering angle shows a single effective diameter of around 11.6 nm.
As shown in Figure 3, stable monomeric monoclonal antibody can be measured effectively at a single scattering angle, θ = 90o (Figure 3). This yields an effective diameter of around 12 nm, and a low, near zero, polydispersity. The correlation function (Figure 3: main panel) is best described by a single exponential decay, and no aggregate is detected.
In contrast, chemically stressed antibody (Figure 4) cannot be described by a single decay, nor can it be meaningfully described as a continuous distribution of particle sizes. It is clear that, in this case, this sample contains multiple discrete, and well-defi ned populations (Figure 4: lower panel). Since these Γ’s span multiple orders of magnitude in s-1, we are able to resolve them only by carefully selecting a correlator layout that covers a wide range of delay times (high, medium and low -speed channels).
Conclusion
The above described method and technique clearly show how optimisation improves DLS results particularly for challenging applications such as proteins mostly are. The fl exibility of a goniometer system coupled with an easily confi gurable correlator, offer the best possible platform for this type of investigation. Such systems provide a proven pathway to extraction of valuable information out of a complex sample consisting of native and aggregated proteins.
Figure 5. Resolvability of multimodal distribution as a function of scattering angle, θ, and scattering vector, q2. Careful choice of both correlation layout, Γ(τ), and scattering angle allow for monomer to be measured with a high degree of precision, even in the presence of heavily aggregated protein.
It is also apparent that our choice of scattering angle (Figure 5) benefi ts us as well, since these rates are only fully resolvable as we approach higher angles. This is in addition to our I(q,τ) ~ d6 dependence, alluded to above.
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