I am studying a protein that is co—localized with The plasma membrane. I want to understand if it binds directly to it or via an adaptor. I want to use fluorescent microscopy and in vitro assays to test it. Can someone help me please? thank you in advance
I think the first thing you should do is to look at the structure of the protein: look for hydrophobic surfaces or domains that are known to interact with other components of the cell. This can provide some information on what domains might interact with the plasma membrane. You could design a few protein truncations, express them ectopically and use microscopy to see if it still localises at the plasma membrane. - This will tell you what domains are responsible for the interaction.
You could do a membrane flotation assay to see if your protein interacts with lipids and/or treat the cells with methyl-beta-cyclodextrin (that removes cholesterol from the membrane) and see if your protein is associated with lipid rafts.
Ultimately you could use tandem affinity purification/immunoprecipitation to pull down interaction partners and use mass spectrometry to identify any potential adaptors. If something comes up, you can knock-down the adaptor protein and visualise by microscopy if the protein still localises at the membrane or not.
A protection assay may help with this.
https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.ncbi.nlm.nih.gov/pubmed/25939013&ved=0ahUKEwisv-6t6o7XAhWBWrwKHXs6CgoQFgguMAM&usg=AOvVaw1g6dZ2UUszd4jo2N9fBNp8
https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018527/&ved=0ahUKEwisv-6t6o7XAhWBWrwKHXs6CgoQFggpMAI&usg=AOvVaw3rwFfkBOX1DMtFKN_mMDkE
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