Hello everyone.
I want to confirm protein-protein interaction using the PLA assay in human cd4-t-cells.
I would be very happy on any experience regarding how good it works, choice of antibody and whether to go for a direct or indirect test (labeled primary or secondary antibody), also how good the protocol works with cytospin.
I am thankful for any suggestion.
Best
Christian
I am aware the attached paper. I hope it helps. Best of luck.
I made a couple of tests on epithelial cell lines and, in my hands, the results really depend on the context. I mean, the technology is potentially very interesting but the crucial point is the availability of specific primary antibodies against the target proteins. A certain degree of "background signal" exists, so it is also necessary to have good negative controls to compare the obtained results (for instance, a mutated protein lacking the putative interaction domain, etcetera...). However, in the right conditions this approach could be very informative! Thus, you would probably have to make some tests in your system and see what is coming out... Good luck!
Hi Christian,
We published by December 2016 a very detailed protocol to use PLA in cells, mice and human tissue. In additon, we show how to count and improve the signal (deconvolution) and troubleshooting. You can find the protocol here http://onlinelibrary.wiley.com/doi/10.1002/cpph.15/full
Best,
Salvador
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