Phalloidin staining on the test and control cells show difference in organisation of polymerised filamentous actin. Can we show this difference in the extent of polymerisation or in the amount of polymerised actin through SDS-PAGE followed by western blotting.
You can monitor it by measuring the soluble and insoluble fraction in F-actin stabilizing lysis buffer conditions. For a commercial option, there are kits available: For example.
http://www.cytoskeleton.com/pdf-storage/datasheets/bk037.pdf
I had also tried it long time back, looking at protocols from a book available via google bookshelf. Dont remember the right source, but the main component in the lysis buffer was Imidazole.
Good Luck
If these are mammalian cells from tissue culture - use a cell permeabilization buffer containing (tris/salt and the usual suspects) 0.32M Sucrose and 0.5% NP-40. The time of incubation can be varied to allow soluble contents to leak into solution and then actin can be deferentially quantified in Supernatant versus pellet using SDS page (as the kit recomments). Remember to do the assay on ICE and if need be add actin stabilizers.
Thank you Dr. Sen, actually we do not have any kit, please suggest if we can do it without kit..
Yes you can do it without Kit too .... all you have to do is quantitatively load and run a protein gel for all your samples on the same gel, do a western blot and probe it with an actin antibody and develop it using ECL if possible. The details of such a protocol could be obtained from standard protocol books as Maniatis or Current Protocols in Molecular Biology.
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