Visualize protein phosphorylation events in situ

• Detect phosphorylation status of individual receptors with high specificity
• Quantify modification events by counting discrete fluorescent spots
• Localize post translational modifications in situ

Phospho-specific antibodies are notorious for high levels of non-specific binding to closely related receptors, compromising data from phosphorylation studies. Duolink® overcomes this problem by enabling the use of dual recognition, one primary antibody against the target protein and one primary antibody against the specific phosphorylated site. This, together with the signal amplification of Duolink, provides a highly specific method for signal transduction studies. In this example, visual and quantifiable data is obtained after assaying stimulated BJ h TERT cells for phosphorylated PDGF receptor ß.

Phosphorylation of PDGFR ß

PDGF receptor ß is a transmembrane protein tyrosine kinase. Upon activation by the growth factor PDGF-BB, these receptors dimerize and become autophosphorylated at numerous sites. This protein modification activates several signaling pathways that promote cell proliferation, motility and survival. Jarvius et al. found that the phosphorylated isoforms of the PDGFR ß were difficult to distinguish using normal immunofluorescence and immunohistochemistry and that Duolink offeres greater selectivity than IF and IHC and generates countable fluorescent signals.

Duolink - detect and quantify phosphorylation

Traditional cell imaging methods like immunohistochemistry (IHC), immunocytochemistry (ICC) and immunofluorescence (IF) can exhibit limited sensitivity and selectivity when studying post translational modifications in situ, and observations are only semi-quantitative at best. There has long been a need for better methods to objectively quantify protein phosphorylation in signal transduction studies, as well as to visualize the modifications in situ, at naturally occurring levels.

Duolink resolves these limitations by providing sensitive means to detect and visualize not only protein modifications, but also individual proteins and protein interactions, in situ. Importantly, Duolink allows discrete detection of endogenous proteins at physiological expression levels and reveals both their subcellular location and the relative frequency of occurrence.

For the Duolink in situ Proximity Ligation Assay, any pair of primary antibodies can be used, together with secondary antibody labeling. These secondary antibodies, or Duolink PLA™ probes, contain unique DNA strands capable of triggering a DNA amplification reaction when the secondary antibodies are in close proximity (<40 nm). The amplification product is subsequently highlighted using fluorescent oligonucleotide probes, resulting in the generation of one discrete fluorescent spot for each single, modified protein. Spots are counted in a standard fluorescence microscope and image analysis software enables the result to be digitally quantified, displayed and documented. No modification of cells or tissues is required beyond fixation using standard methods.

Selection and qualification of primary antibodies

The PLA probes in the Duolink kit are secondary anti-species antibodies for detecting presence of analyte-specific primary antibodies. It is advisable to verify the function of primary antibodies in combination with selected sample, fixation and retrieval methods in a standard IHC, ICC or IF staining experiment before setting up the Duolink assay. The assay can be greatly improved with respect to countable signals by testing different concentrations (titers) of primary antibodies and PLA probes. This can be achieved by running a Duolink Q assay. Appropriate blocking solution and control samples will further ensure optimal result. Depending on the protein or interaction being assayed, it may be important to evaluate the choice of epitopes that are available for labeling.

Visualizing endogenous levels of phosphorylated PDGFR ß

Duolink generates a discrete fluorescent signal for each detected modification, which facilitates an objective approach to image analysis and comparison of phosphorylation events in untreated and PDGF-BB stimulated cells (Fig. 2).

With the BlobFinder Image Analysis software, the relative amounts of phosphorylated receptors can be quantified in each single cell (Fig. 3).

Material and methods

As for any method used to detect specific molecules in cells and tissues, special considerations must be taken that may be unique to your particular material and analyte, to make them amenable to treatment and detection. You may need to optimize analyte-specific aspects such as the permeability of cellular compartments and the availability of epitopes through fixation, and the elimination of non-specific background by the use of Duolink blocking reagents. You may have to optimize the antibody titer for both primary antibodies and PLA probes

Immortalized human foreskin fibroblasts (BJ hTert) were seeded on glass slides in minimum essential medium (MEM supplemented with 10% FCS in the presence of antibiotics and L-glutamine). After 24 h the cells were starved over night in medium containing 0.5% FCS. Then the cells were stimulated with 100 ng/ml human PDGF-BB for 1 h on ice, rinsed once with icecold phosphate-buffered saline (PBS) and fixed for 1 h in ice-cold 70% ethanol. Subsequently, slides were air dried and stored at -20 °C until analyzed with the Duolink assay according to the Custom blocking solution protocol in the Duolink User Manual (available at www.olink.com). For primary antibodies, 20% goat serum was found to be the optimal blocking reagent.

Image acquisition was done at 20X magnification (Zeiss Plan-Apochromat, 20X/0.8NA dry) with the Zeiss Axio Imager M1 fluorescence microscope and the AxioVision 4.5 software (Carl Zeiss). Images were captured with an AxioCam MRm-camera with an extension tube of 1X (Zeiss 60NC 1” 1x). Filters used were 575/605 nm (in situ PLA signal) and 350/461 nm (nuclear stain). A Z-stack of 20 images was taken at 20X magnification of the in situ PLA image channel. One image of nuclei in the Hoechst/DAPI channel was aquired by auto-exposure and auto-focus. Typically, an image field contained 25-30 cells. All images were treated equally and exported to TIFF format for subsequent analysis in the BlobFinder image analysis software (Allalou A. and Wählby C., BlobFinder Image Analysis software: www.cb.uu.se/~amin/BlobFinder).

References

Söderberg O., Gullberg M., JarviusM., Ridderstråle K., Leuchowius K-J., Jarvius J., Wester K., Hydbring P., Bahram F., Larsson L-G., Landegren U. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nature Methods 2006 Dec;3(12):995-1000

Jarvius M., Paulsson J., Weibrecht I., Leuchowius K-J., Andresson A-C., Wählby C., Gullberg M., Botling J., Sjöblom T., Markova B., Östman A., Landegren U., Söderberg O. In situ detection of phosphorylated PDGF receptor ß using a generalized proximity ligation method. Mol Cell Proteomics. 2007 Sep;6(9) 1500-1509