Proximity Ligation Assay To Detect Protein Interactions

February 06, 2017

Colorado State University
Food Science and Human Nutrition
Graduate Student

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Company:

Sigma-Aldrich

Product Name:

Duolink® In Situ Red Starter Kit Mouse/Rabbit

Catalog Number:

DUO92101

The purpose of this experiment was to test and quantify mitochondrial associated membrane (MAM) formation using two proteins that have been shown to interact when the MAM is formed. The MAM is an important point of contact between the mitochondria and ER and has been shown to be involved in nutrient and energy transfer between the two organelles. VDAC is a protein that is typically found in the mitochondria while IP3R is found in the ER. The goal of this experiment was to test the interaction between VDAC and IP3R using this proximity ligation assay as a surrogate measure of MAM formation.

Experimental Design and Results Summary

Application

Fluorescent Microscopy

Starting Material

Fixed and permeabilized H4IIE rat hepatoma cells

Protocol Overview

The directions that come with this kit are very easy to follow and broken down so there are no confusing steps. Once cells are fixed and permeabilized, primary antibodies are incubated on the cells in the antibody diluent the kit provides. PLUS and MINUS probes are then incubated on the cells which act as the secodary antibody, binding to their respective primaries. If the proteins are less than 40nm from one another (representing interaction), the probes will hybridize. The cells then go through a ligation step that completes the circular DNA strand created by the probes. This signal is then amplified with an amplification solution and incubation. This solution also contains the fluorophores that hybridize to the amplified oligo strand and are viewable under a fluorescent microscope. Using the solutions and wash buffers that this kit provides, it is a relatively simple procedure.

Tips

-We used super glue to glue our coverslip to a glass slide before going through the PLA. Make sure to seal the edges so all the different treatments don't run off the cells. Also, we initially thought that the recommended volume was way too little. However, after trying it, it worked very well and covered the coverslip perfectly.

Results Summary

This assay worked the first time it was tried. As a negative control, we used a single primary antibody instead of both and saw nothing, which was great. The only problem was the background signal was relatively high, making the dots (interactions) hard to distinguish and quantify. This assay really stresses on the wash steps and this could be a reason for high background among other things. Overall, happy with the results, but this will take some optimization to get ideal results without a lot of background.

DOI or PMID #

N/A

Additional Notes

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Summary

The Good

Kit comes with everything you need including directions, troubleshooting and all solutions to get through the procedure. All you need are cells that have been fixed and permeabilized. Assay will work first try if all reagents from the kit are used correctly and directions are followed

The Bad

High background signal. May have to start tweaking the concentrations of antibodies, probes, ligation, amplification solutions. Could take a lot of time and resources

The Bottom Line

This kit is awesome for testing the interaction between two different proteins. I think it is a really useful tool for biochemistry labs interested in protein interactions. It's easier to perform than FRET and nice to actually see little dots of interaction under a microscope. The analytical side of things are a little more tricky, especially if your microscope camera isn't top of the line. Will continue performing experiments until ideal conditions are met.

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