I would have a few questions regarding isolation of proteins localized in plasma membrane and/or cytoskeleton.
The protein of my interest should be anchored in plasma membrane (it should pass membrane by several domains). However, when I do subcellular fractionation using different commercially available kits, I always detect the protein in cytoskeleton and never in any membrane fraction. Or when I perform separation of triton x-100 soluble-insoluble fraction, the protein is in triton insoluble fraction (cytoskeletal proteins). A subcellular localization using immunofluorescence microscopy I can not do, since antibodies against this protein are not very specific (I detect many additional bands).
1. is it possible that protein is really anchored in plasma membrane/passing plasma membrane, but because it also interacts with some cytoskeletal proteins it is always in cytoskeleton fraction?
2. there are structures called focal adhesion in cell - proteins connecting cytoskeleton with extracellular matrix. May be the protein interacts in plasma membrane with focal adhesion proteins. If I would separate cytoskeleton proteins (e.g. using triton x-100) the focal adhesion proteins would be mainly in triton insoluble (cytoskeleton) or soluble fraction?
3. Do you have any ideas, how I can get to know whether the protein is really in plasma membrane?
Thanks in advance.
Isolation of plasma membrane, focal adhesion and cytoskeleton proteins
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