In Situ Proximity Ligation Assay (PLA) Kit components: A Comprehensive Review

May 10, 2016

Purdue University
Animal Sciences
Postdoctoral Fellow

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Company:

Sigma-Aldrich (formerly a product of Olink Bioscience)

Product Name:

Duolink in situ proximity ligation assay (PLA) kit

Catalog Number:

DUO82049-4L, DUO92014-100RXN, DUO92002-100RXN, DUO92006-100RXN

The aim of my research was to detect if 14-3-3η protein interacts with α-tubulin at the meiotic spindle of eggs during mouse oocyte maturation. The project was conducted as follow up on results showing accumulation and co-localization of the 14-3-3 isoform at the meiotic spindle of mouse eggs matured in vivo as well as in vitro, along with disruption of the spindle asembly upon microinjection of translation-blocking morpholino oligonucleotides targeting the 14-3-3 isoform. I performed the Duolink in situ proximity ligation assay (PLA) to determine if 14-3-3η interacts directly with α-tubulin in the meiotic spindle during mouse oocyte maturation. The PLA has been reported to have been effectively used to not only detect protein-protein interactions at the single molecule level directly within individual cells, but also for visualization and relative quantification of the intracellular sites of the interactions in different types of cells and tissues. The PLA technique is sensitive, specific, and provides a high signal to noise ratio because the signal is amplified and close proximity of the target proteins is required. Thus, the method permits detection of two proteins that interact at the molecular level.

Experimental Design and Results Summary

Application

Visualization and relative quantification of in situ protein: protein interaction within single cells and tissues, at the single molecule level.

Starting Material

Primary cells and tissues, freshly isolated (or cultured) and fixed.

Protocol Overview

In the PLA method, specific primary antibodies (raised in different species) bind to target proteins. A pair of oligonucleotide-conjugated secondary antibodies (PLA probes) bind to the primary antibodies and when the PLA probes are in close proximity.

Tips

Appropriate controls should be used, both postitive and negative, along with biological as technical controls. Need to optimize incubation times and reaction volumes depending on the cell type or tissue used, and depending on whether the assay is performed on slides or in glass-bottom wells / dishes.

Results Summary

The in situ PLA revealed some interactions (noted by distinct bright fluorescent reaction spots) of 14-3-3? with a-tubulin throughout the cytoplasm of all mouse eggs matured in vitro, along with a prominent accumulation of the interaction sites at the meiotic spindles in 16 out of 18 eggs examined. In addition, an abundance of the interaction sites was noted along the cell cortices next to the spindles of those eggs studied. These observations indicate that 14-3-3? interacts with a-tubulin in mouse eggs and that such interactions are dramatically more prevalent in the region of the meiotic spindle as would be predicted by the colocalization and enhanced concentration indicated by the standard immunofluorescence experiments.

Additional Notes

None

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Summary

The Good

Sensitive and fast, yet effective and convenient technique requiring few cells! Can detect even weak or transient interactions between proteins.

The Bad

Long incubation times and multiple controls to be used in order to rule out possibilities of false positive results.

The Bottom Line

Great product which can be used with confidence for supporting, confirming and validating standard biochemical results of protein-protein interactions like co-IP, GST pulldowns etc.

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