Protein microarrays, on which thousands of discrete proteins are printed, provide a valuable platform for functional analysis of the proteome. They have been widely used for biomarker discovery and to study protein-protein interactions. The accomplishments of DNA microarray technology, which had enabled massive parallel studies of gene expression, sparked great interest for the development of protein microarrays to achieve similar success at the protein level. Protein microarray detection techniques are often classified as being label-based and label-free. Most of the microarray applications have employed labelled detection such as fluorescent, chemiluminescent and radioactive labelling. These labelling strategies have synthetic challenges, multiple label issues and may exhibit interference with the binding site. Therefore, development of sensitive, reliable, high-throughput, label-free detection techniques are now attracting significant attention. Label-free detection techniques monitor biomolecular interactions and simplify the bioassays by eliminating the need for secondary reactants. Moreover, they provide quantitative information for the binding kinetics. In this article, we will review several label-free techniques, which offer promising applications for the protein microarrays, and discuss their prospects, merits and challenges.

a b s t r a c tFaithful segregation of chromosomes during mitosis and meiosis is the cornerstone process of life. Cohesin, a multi-protein complex conserved from yeast to human, plays a crucial role in this process by keeping the sister chromatids together from S-phase to anaphase onset during mitosis and meiosis. Technological advancements have discovered myriad functions of cohesin beyond its role in sister chromatid cohesion (SCC), such as transcription regulation, DNA repair, chromosome condensation, homolog pairing, monoorientation of sister kinetochore, etc. Here, we have focused on such functions of cohesin that are either independent of or dependent on its canonical role of sister chromatid cohesion. At the end, human diseases associated with malfunctioning of cohesin, albeit with mostly unperturbed sister chromatid cohesion, have been discussed.

SummaryMeiosis is a specialized cell division process through which chromosome numbers are reduced by half for the generation of gametes. Kinetochore, a multiprotein complex that connects centromeres to microtubules, plays essential role in chromosome segregation. Ctf19 is the key central kinetochore protein that recruits all the other non-essential proteins of the Ctf19 complex in budding yeast. Earlier studies have shown the role of Ctf19 complex in enrichment of cohesin around the centromeres both during mitosis and meiosis, leading to sister chromatid cohesion and meiosis II disjunction. Here we show that Ctf19 is also essential for the proper execution of the meiosis I specific unique events, such as non-homologous centromere coupling, homologue pairing, chiasmata resolution and proper orientation of homologues and sister chromatids with respect to the spindle poles. Additionally, this investigation reveals that proper kinetochore function is required for faithful chromosome condensation in meiosis. Finally, this study suggests that absence of Ctf19 affects the integrity of meiotic kinetochore differently than that of the mitotic kinetochore. Consequently, absence of Ctf19 leads to gross chromosome missegregation during meiosis as compared with mitosis. Hence, this study reports for the first time the differential impact of a non-essential kinetochore protein on the mitotic and meiotic kinetochore ensembles and hence chromosome segregation.

In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12–0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast.