I am confident and reasonably competent with both immuno-staining and co-IP. My immunofluorescent co-staining indicates that my protein of interest does bind the actin cytoskeleton.
I have been able to co-IP my protein of interest with binding partners that others have reported. I have not been able to co-IP my protein with actin.
I suspect that the conditions that allow efficient extraction of actin from its filamentous form will likely disrupt protein protein interactions necessary to co-IP.
There may be a simple condition or reagent (actin de-polymerizing reagent?) that would allow a fairly straight forward co-IP with actin.
I want to be very open here for any suggestions of techniques that would demonstrate that my protein of interest binds the actin cytoskeleton. I suspect that there may be acellular assays and even kits for this kind of thing, but I have not done this.
Hi Neal,
CoIP is one of the techniques that can be used but there are some others.
For example with fluorescent proteins you can do FRET experiments or Bimolecular Fluorescent Complementation.
There are several manners to do FRET and you should get in touch with some Imaging Facility (I guess there is at least one in Seattle).
BiFC is quite simpler but can produce false positives.
If your antibodies work well for immunostaining, you could also try to see you antigen by Electron Microscopy (TEM). Primary antibodies are revealed by antibodies coupled to gold beads. You can measure the distance between the gold beads and the microfilament.
There is also Yeast Two-Hybrids that can help you, but I'm not specialized in that technique.
Cheers
Oliv
This kit from Cytoskeleton may be used on cell lysates with overexpression. https://www.cytoskeleton.com/bk001#:~:text=The%20actin%20binding%20protein%20spin,using%20a%20spin%20down%20method.
If you want there are various in vitro experiments, where one can use recombinant purified proteins (both actin and protein of interest). If you are interested in this approach I can suggest many experiments. I will elaborate on them further, based on your response.
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