Affimers are a good alternative to antibodies — they’re small engineered binding proteins that possess the desirable properties of antibodies, such as high specificity and affinity, while avoiding some of the problems commonly associated with antibody use. Recently, Affimers were used to detect atypical ubiquitin (Ub) chains, a target that has been understudied because of the lack of tools to enable their detection. MicroScale Thermophoresis (MST) proved to be essential in the characterization of these Affimers, relieving the limitations of alternative technologies in obtaining binding affinity data.
Little is known about the biological function of atypical ubiquitin (Ub) chain types, such as K6, K11 and K33, because tools and techniques to detect them are scarce. One reason for this is that Ub-specific antibodies are very difficult to generate due to the high identity of Ub between species, thus linkage-specific antibodies have been generated for only five of the eight Ub chain types. In a way to push past this limitations, Michel et al. described linkage-specific Ub Affimers to both K6 and K33 linkages, opening the door to the otherwise impossible study of these atypical ubiquitin chains.
The binding functionality details of these linkage-specific Ub Affimer proteins was analyzed by several technologies. According to isothermal titration calorimetry (ITC)- both K6 and K33 Affimers bound tightly (<1 nM) to their targets without cross-reacting with one another. Nevertheless, accurate binding constant values (Kd) cannot be obtained due to the detection limit of this technique. Qualitative kinetic analysis by surface plasmon resonance (SPR) on the other hand, demonstrated that linkage specificity is achieved through very low off rates only for the cognate ubiquitin chain. Interestingly, under these low off rates conditions, measurement of Kd values is not straightforward by SPR either. Taking advantage of the high sensitivity and ease of use of Microscale Thermophoresis, Kd values were obtained for engineered versions of the K6 and K33 Affimers, revealing binding affinities in the pM range towards their cognate Ub with negligible affinity for the other Ub linkages. Moreover, it showed that the K33 Affimer cross-reacts with K11 linked chains with a low nM binding affinity.
The performance of these characterized Affimers was further demonstrated in a number of applications such as western blots, pull-downs, mass spectrometry and confocal microscopy. These applications in conjunction with several technology tools allowed researchers to gain insight into the significance of atypical ubiquitin chains in the cellular context.
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