We have been trying to examine tyrosine phosphorylation-based signalling and protein-protein interactions in macrophages. Since our traditional IP lysis buffer contains NP-40, we may be missing some important protein-protein interactions at the plasma membrane. To examine which phosphoproteins signal from a RAFT platform, we tried a detergent-free Optiprep RAFT isolation protocol following the method of MacDonald & Pike (2005) but our protein concentrations were extremely low. Does anyone have a good, relatively simple RAFT protocol that they have used successfully in macrophages?
Thanks Aaron. It seems that detergents can produce significant artifacts in raft preps but I think Triton X may be the way to go for the moment to improve protein concentrations.
Hello Fiona,
We have some work described in: M. Garcia-Marcos, S. Pochet, S. Tandel, U. Fontanils, E. Astigarraga, J.A. Fernandez-Gonzalez, A. Kumps, A. Marino and J.P. Dehaye
Characterization and comparison of lipid rafts isolated by two different methods from rat submandibular gland cells. : B.B.A., Biomembranes, 1758, 796-806, 2006. May be it could help you. Greetings!!. Aida
Hi Aida. Thanks for that reference. It looks very interesting. I think we will try a detergent extraction and bear in mind that it could affect partitioning of some proteins. Cheers,
Fiona.
fiona,
biochemically, lipid rafts have been defined as detergent-resistant membranes (DRM).
some paper showed that oncogenic tyrosine kinases are detected in the DRMs. see below. probably, even using detergents, you can do co-IP some proteins in the DRM.
Iwabuchi K, Nagaoka I. Blood. 2002 Aug 15;100(4):1454-64.
Nakayama H, Yoshizaki F, Prinetti A, Sonnino S, et al.,
J Leukoc Biol. 2008 Mar;83(3):728-41. Epub 2007 Nov 30.
thank you,
kaz
Thanks for your input and references, Kaz. We've actually stuck with our detergent-free protocol and just acetone precipitated the fractions to increase protein concentrations at the moment. If this isn't sufficient, we'll turn to some of the suggestions you and other have made.
Cheers,
Fiona.
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