I am performing a colocalization analysis of two proteins. I already have confocal microscopy images of my proteins of interest, but I am confused about how to measure the degree of colocalization of the fluorescence signals. I know that there are several colocalization indexes that I can use, but I do not fully understand howto interpret them and which are the best for my research questions. Can anybody please help me or provide some reading suggestions about this topic? Thanks!
Please see the JACOP plugin for ImageJ as well as its associated review (http://rsbweb.nih.gov/ij/plugins/track/jacop.html) for an in depth overview of this technique. My favorite is the van Steensel spatial correlation method which has similarities to the image cross correlation methods that Paul Wiseman has developed. We used this method extensively in our recent paper on yeast microdomain colocalization and antilocalization (http://www.nature.com/ncomms/journal/v4/n1/full/ncomms2370.html?WT.ec_id=NCOMMS-20130122).
So you can use a FRET (Förster resonance energy transfer) so you have both Proteins labeled. An only if they are in connection or in the near you can see Fluorescence intensity.
Or what do you mean?
The following article may be useful
http://www.olympusmicro.com/primer/techniques/confocal/applications/colocalization.html
Good luck!
Please see the JACOP plugin for ImageJ as well as its associated review (http://rsbweb.nih.gov/ij/plugins/track/jacop.html) for an in depth overview of this technique. My favorite is the van Steensel spatial correlation method which has similarities to the image cross correlation methods that Paul Wiseman has developed. We used this method extensively in our recent paper on yeast microdomain colocalization and antilocalization (http://www.nature.com/ncomms/journal/v4/n1/full/ncomms2370.html?WT.ec_id=NCOMMS-20130122).
The image analysis and FRET are excellent choices to study two protein colocalization. In addition, in our lab, we also used mass-spectrometry to study the proteins colocalization.
If you are looking for something a little more specific try switching to a proximity ligation assay (PLA, try Olink - Duolink) they explain it on their website basically just changes the secondary detection. Proteins (labelled with antibodies) only fluoresce if less than 40nm apart - thus if its an on/ off interaction you can measure it as an interaction more specifically than just fluorescent overlap which can be very difficult to interpret in some systems especially if proteins are ubiquitously expressed.
Hope this helps,
S
@Scott- have you used PLA? I agree it could be a useful assay here, but the community of experienced users to help troubleshoot the method is still quite small.
Yep, we have used it multiple publications Nature, Circ Res etc etc . There is almost no optimisation. Just buy the kit, treat your cells/ sections as you would normally through to the end of the primary antibody detection part, then follow the instructions for secondary detection (PLA part). We have used it with several protein complexes as have others I work with and it is about one of the easiest switches out there. Very clear results (if a complex forms). always happy to help with it but I would say you wouldn't need it.
I have used the duolink kit successfully as well with my own first and secondary antibody incubation buffer, but had to add carrier DNA to it to get consistent results.
Yes--the plugin will work with all types of images. Of course, the lower your z resolution is, the harder colocalization becomes (greater probability of objects above and below one another overlapping). With structured illumination, be very careful that your reconstruction doesn't have ringing patterns that are present in both images. These will correlate strongly especially for the van steensel method.
sure thing and just to update (Daniel pointed this out. The Duolink kit I used is now distributed through Sigma: http://www.sigmaaldrich.com/life-science/proteomics/proteomics-products.html?TablePage=112232138
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