Abstract
Quantitative determination of protein thermodynamic stability is fundamental to many research areas, both basic and applied. Although chemical-induced denaturation is the gold-standard method, it has been replaced in many settings by semi-quantitative approaches such as thermal stability measurements. The reason for this shift is that chemical denaturation experiments are labour-intensive, sample-costly and time-consuming, and it has been assumed that miniaturisation to a high-throughput format would not be possible without concomitantly comprising data quality. Here we exploit current technologies to create a high-throughput label-free chemical denaturation method that is capable of generating replicate datasets on multiple proteins in parallel on a timescale that is at least ten times faster, much more economical on sample, and with the potential for superior data quality, than the conventional methods used in most research labs currently.
Highlights
We exploit the capabilities of the latest generation of commercially available plate readers combined with a small-scale protein purification procedure to produce a method for high-throughput quantitative measurement of protein stability
The rapid high-throughput approach we describe here incorporates the plate-reader measurement into a protocol for small-scale protein expression and purification, enabling multiple technical and biological replicates of protein stability to be acquired in parallel on a very short timescale: 2 days from cell transformation to obtain data on 12 different proteins
We develop a protocol that combines this measurement with a rapid small-scale protein purification method, thereby streamlining the whole process and dramatically decreasing the timescale of such experiments and increasing the number of proteins that can be characterised in parallel
Summary
We exploit the capabilities of the latest generation of commercially available plate readers combined with a small-scale protein purification procedure to produce a method for high-throughput quantitative measurement of protein stability. Plate-reader sensitivity in the UV range has improved tremendously in recent years, and we demonstrate that it can provide a very fast and accurate method for chemical denaturation measurements on most proteins It is extremely economical on sample compared with conventional fluorimeter-based methods, thereby obviating the need for the very time-consuming process of purifying proteins on a large-scale from litres of E. coli culture. The plate-reader approach means that we avoid the time-consuming and labour-intensive process of sample equilibration Performing these types of replicates using the conventional fluorimeter instrumentation and the standard large-scale purification methods that are needed to generate sufficient protein for this instrumentation makes the acquisition of a single denaturation curve a labour-intensive and time-consuming task that lacks any real scope for parallelisation
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