Hello,
I work on a viral protein which is anchored on the membrane due to palmitate residues and an alpha helix. Therefore, it is strongy membrane associated.
I have managed to perform cytoplasm/membrane fractionation assays, and have observed that this protein is significantly found in the membrane fraction.(P15) Therefore, there is a significant enrichement of this protein on the membrane fraction.
The idea is to be able to perform immunopreciptation(GFP trap-Chromtek) of this protein on the membrane fraction, and then to perfrom mass spec.
The question is: What is best buffer in order to resolubilize the membranes in order to successfully perform GFP-trap? Preferrably it would be a buffer that solubilizes the membrane proteins allowing IP without losing partners.
Thank you for your help
Any clarification..you can ask!
Hi, there's no one right answer to this question. I can suggest some reading from our research that will inform you ideas about how to go about this. We mostly work on soluble proteins, but we have used these methods for nuclear membrane, ER, and plasma membrane protein complexes with success. The references in these articles include those that are instructive about detergents etc. The basic idea is - one has to search around the reagent space to find something that works well and one can't really know that in advance at our present state of understanding. Of course, educated guesses based on the biochemical properties of your target protein can (and should) be made.
LaCava, J., Fernandez-Martinez, J., Hakhverdyan, Z. & Rout, M. P. Protein Complex Purification by Affinity Capture. in (eds. Andrews, B., Boone, C, Davis, T & Fields, S.) vol. 2016 382–397 (Cold Spring Harbor Laboratory Press, 2016).
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LaCava, J. et al. Affinity proteomics to study endogenous protein complexes: Pointers, pitfalls, preferences and perspectives. BioTechniques 58, 103–119 (2015).
LaCava, J., Jiang, H. & Rout, M. P. Protein Complex Affinity Capture from Cryomilled Mammalian Cells. Journal of Visualized Experiments e54518 (2016) doi:10.3791/54518.
Hakhverdyan, Z. et al. Rapid, optimized interactomic screening. Nature methods 12, 553–560 (2015).
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