Detect, Visualize and Quantify Single Post-Translational Modifications

Caleb Hopkins

Duolink^® In Situ products enable detection, visualization, and quantification of protein events in tissue and cell samples prepared for microscopy. The in situ PLA^® technology, on which these products are based, offers advantages over traditional procedures, as selectivity is increased with dual recognition of target protein(s) by antibody probes, a signal can only be generated when the antibody probes are bound in close proximity, and DNA amplification results in increased sensitivity.¹

The in situ PLA^® technology is designed to determine the proximity of two epitopes using primary antibodies specific to each epitope.^2,3 An antibody complex with conjugated oligonucleotides forms at each epitope. A ligation reagent is then added and, if the two epitopes are in close proximity, the conjugated oligonucleotides will hybridize to a connector oligonucleotide.^1,3 The formation of a circular DNA molecule serves as a template for a rolling circle amplification (RCA) reaction.^1-3 After the RCA reaction, appropriately labeled detection oligonucleotides (fluorophores for fluorescence and horseradish peroxidase [HRP] for brightfield microscopy) are added, resulting in hybridization of 500–1,000 labeled detection oligonucleotides to the repeated sequence encoded in the DNA circle, allowing observation of a single protein event.

Protein events that can be detected by in situ PLA^® studies include protein post-translational modifications (PTM), protein-protein interactions, and protein expression.

Research in signal transduction has led to a better understanding of the mechanisms that control cellular processes such as proliferation, migration, and apoptosis.^1,2 PTM, such as phosphorylation, ubiquitination, acetylation, and glycosylation, of pathway proteins are integral control components to these processes.^1,2 A comprehensive understanding of a signaling cascade requires knowledge of the spatial and temporal dynamics of these post-translational modifications. This information is not always obtained through traditional methods. Recently, in situ PLA^® experiments have uncovered PTM in both cell line models and formalin-fixed/paraffin-embedded tissues that were overlooked in studies using other procedures.¹

The phosphorylation state of signaling pathway enzymes is critical for enzymatic activity and an in situ PLA^® study can be used for detection of phosphorylation events.^1,3 Using separate primary antibodies against the target protein and the phosphorylation site, measurements of changes in cellular response can be determined unlike current diagnostic methods that measure expression levels of individual proteins. A recent article describes methods for determining melanopsin signaling.³ It was found that signaling is regulated by the phosphorylation of serine and threonine residues on intracellular loops 2 and 3 by protein kinase A.³ Antibodies against the carboxy tail of melanopsin and against phosphoserine were used in conjunction with oligonucleotide conjugated secondary antibodies (PLA^® probes).³ Addition of fluorescently-labeled oligonucleotide probes specific for the amplified DNA (Duolink^® In Situ Detection Reagent) resulted in a distinct, fluorescent spot for each pair of interacting antibody molecules.

In another article, quantitative expression levels of isoform-specific activation (phosphorylation) of Akt1 and Akt2 in cell lines and breast cancer tumor tissue is described.⁴ The amount of activated Akt was measured to determine the correlation between activated Akt1 and Akt2 isoforms with breast cancer outcomes.⁴ Interestingly, previous immunohistochemistry data did not compare with the phosphorylated Akt1 or Akt2 measurements obtained with the in situ PLA^® method, suggesting in situ PLA^® experiments with Duolink^® In Situ reagents may provide information that cannot be obtained by standard assays.⁴

Variation of in situ PLA^® procedures allows for detection of protein events beyond PTM. Co-immunoprecipitation, Western blot, crosslinking, and pull down assays traditionally have been used to validate, characterize, and confirm protein-protein interactions; however, these procedures have limitations. The proximity requirement of the in situ PLA^® reaction would be ideal for detection of protein-protein interactions even at endogenous expression levels, utilizing separate primary antibodies against each target protein.

Future endeavors for in situ PLA^® studies include a focus on the presence and activity of low-abundance proteins, multiplexed assays to generate biomarker-profiles for diseases, and pharmacologic investigations addressing efficacy and safety concerns for use in clinical trials.¹

For more papers, protocols, and products related to Duolink^® visit sigma.com/duolink.

References

1. Weibrecht I, Leuchowius K, Clausson C, Conze T, Jarvius M, Howell WM, Kamali-Moghaddam M, Söderberg O. 2010. Proximity ligation assays: a recent addition to the proteomics toolbox. Expert Review of Proteomics. 7(3):401-409. https://doi.org/10.1586/epr.10.10

2. Jarvius M, Paulsson J, Weibrecht I, Leuchowius K, Andersson A, Wählby C, Gullberg M, Botling J, Sjöblom T, Markova B, et al. 2007. In SituDetection of Phosphorylated Platelet-derived Growth Factor Receptor ? Using a Generalized Proximity Ligation Method. Mol Cell Proteomics. 6(9):1500-1509. https://doi.org/10.1074/mcp.m700166-mcp200

3. Blasic JR, Brown RL, Robinson PR. Phosphorylation of Mouse Melanopsin by Protein Kinase A. PLoS ONE. 7(9):e45387. https://doi.org/10.1371/journal.pone.0045387

4. Spears M, Cunningham CA, Taylor KJ, Mallon EA, Thomas JSJ, Kerr GR, Jack WJ, Kunkler IH, Cameron DA, Chetty U, et al. 2012. Proximity ligation assays for isoform-specific Akt activation in breast cancer identify activated Akt1 as a driver of progression. J. Pathol.. 227(4):481-489. https://doi.org/10.1002/path.4022

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