Abstract
Many cellular proteins interact with the cytoskeleton (both actin filaments and microtubules), either dynamically or permanently. This interaction is required during different aspects of the cell life, for example during the process of cell division. In addition, many enzymes interact transiently with actin filaments and microtubules in order to promote their cellular distribution. Several substances with inhibitory capacity can affect this binding and cause damages to cells. This protocol allows to analyze whether a protein interacts with either actin filaments or microtubules and, when applicable, the conditions controlling this interaction.The test is based on the specific binding between the protein of interest and the cytoskeletal filaments. As shown schematically in the diagram of example (see below), the test starts from the cell lysate to which actin filaments (produced from monomeric actin) are added. The mixture (performed under different experimental conditions chosen by the operator) is then incubated so that the protein of interest (in the example, myosin) binds to actin filaments. The sample is then centrifuged in order to separate unbound or weakly-bound proteins from actin filaments to which both the protein of interest and, eventually, traces of less specific proteins are associated.
Keywords: Actin filament, Microtubule, Myosin, Protein binding
Materials and Reagents
Equipment
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Procedure
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Acknowledgments
The protocol was adapted from a previously published paper: Del Duca et al. (2013). The work was supported by PRIN 2007 (grant no. 2007RZCW5S_003) and PRIN 2008 (grant no. 2008BK7RXB), funded by the Italian Ministry of University and Research, and by Bologna University (RFO 2010 [grant no. RFO10DELDU] and RFO 2011 [grant no. RFO11DELDU]) to S.D.D.
References
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Dear colleague, in principle it would be possible but it is practically very difficult to identify the parameters of centrifugation or sedimentation because the cytoskeleton is not a homogeneous compartment, the cytoskeletal elements can have different length, they can form aggregates, therefore having a different mass. it is therefore technically complicated.
Thanks a lot for answering 😉
Dear colleague, I thank you for your question. I think that the polymerized actin can be stored for one day, maybe two at the most. We usually prefer to use it fresh, but I have experienced that after one day the polymerized actin is still intact.bestGiampiero