What would be the best way to study the interaction? Purifying actin and doing on gel binding assays or tagging actin and checking for interaction?
Actin cosedimentation experiments have worked very nicely for us in the past. It is fairly straightforward to do. Here is a brief method taking from one of our papers.
Actin co-sedimentation assays
Muscle G-actin was purified from rabbit skeletal muscle (Pardee and Spudich, 1982) and polymerised in 10 mM Tris, 50 mM NaCl, 100 mM ATP, 1 mM DTT, 1 mM MgCl2, pH 7.0. Assays were performed using 4mM talin and concentrations of F-actin ranging from 0 to 25 mM. The mixture was incubated for 60 min at room temperature and centrifuged at 100 000 r.p.m. for 30 min at 22oC using a Beckman Optima TM ultracentrifuge. Supernatants and pellets were analysed on 12% SDS–PAGE gels, stained using Coomassie blue and scanned. Protein levels in the pellet were determined using Image J software and normalised using talin loading controls (2 mM). Each mutant was analysed in triplicate.
Gingras AR, Bate N, Goult BT, Hazelwood L, Canestrelli I, Grossmann JG, Liu H, Putz NS, Roberts GC, Volkmann N, Hanein D, Barsukov IL and Critchley DR. (2008). The structure of the C-terminal actin-binding domain of talin. EMBO J. 27(2): 458-69
http://www.ncbi.nlm.nih.gov/pubmed/18157087
If you need any further technical details then just ask.
Cheers
Ben
Ben, could you also give me a suggestion on how could I check if my protein interacts with some plant polysaccharides such as cellulose/starch etc? is there an assay for the same?
Hi Madhurima, you can find a description how to measure binding to action by using MicroScale Thermophoresis (MST) in this paper:
"Functional Characterization of Human Myosin-18A and its Interaction with F-actin and GOLPH3"
http://www.jbc.org/content/early/2013/08/29/jbc.M113.497180.short
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