Dear actin experts,
I am trying to reproduce some actin formations in solution but I am not sure of whether what I am observing is polymerised actin or aggregates. Could I please kindly ask you to help me verify from my confocal images (attached) that I am obtaining the desired formations and not just random actin aggregates? Is there a visual difference between actin aggregates and polymerised actin? Please see below a short description of what I believe each picture is presenting.
Picture 1: Thick actin bundle that opens to thinner bundles
Picture 2: “Chunk” of bundles and a thinner bundled formation below them
Picture 3: Meshwork of actin/biotin-actin crosslinked with streptavidin (molar ratio of biotin-labelled actin to actin : 1-50, streptavidin to actin: 1-100)
Picture 4: Thin actin bundles
All the samples contained 1mg/ml of actin (10% rhodamine labelled) and were polymerised in the presence of 50 mM KCL, 2 mM MgCL. 0.2 mM ATP and 10 mM Tris pH 7.5 ( all actin stocks were prepared according to the product datasheets). The sample in picture 4 contained 50 mM of MgCl to increase the yield of the formed bundles.
Harald Birkholz
as this is a section for questions and discussions, what I'm asking is whether there is a distinct visual difference between polymerised actin and aggregated actin. I don't have previous experience in working with actin and in the literature there is a wide range of morphologies that is attributed to polymerisation or aggregation. For example, I've read that the straightness of a formation is a major criterium in order to discriminate polymerised actin from aggregated actin but I've also seen many crumpled structures named as bundles or filaments. Obviously the conditions and the presence of accessory proteins affects these formations and their characterisation. But again, my question to people that have relevant microscopy experience is whether there are distinctive visual differences between these two states. In any case, thanks for answering my question by saying that you cannot answer it.
Hi, not an expert but I believe you have F actin under your condition. Maybe you can add DTT to help further drive the polymerization. If it's just aggregates, it should be like "particle bundles" but F actin gives you "pure line". This article might help you understand it. Thank you.
https://www.nature.com/articles/nchembio755
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