I am extracting proteins from plant samples and am currently attempting to use the TCA/Acetone extraction method. I am having trouble resolubilizing my sample after drying the pellet. I collected the pellet during the extraction process as per the protocol, but I am confused about whether the pellet is the actual plant residue from the initial grinding (using liquid nitrogen) or if it is the extracted protein, since the supernatant is discarded throughout the extraction. I have not yet proceeded with any treatment using DTT/IAA or protein digestion - I simply want to test the sample using a Bradford assay and intend to resolubilize it. If anyone able to answer my questions, it would be very helpful. Thank you
When you do TCA/Acetone precipitation, your protein is as dead as they come. Bradford won't like aggregated proteins, so you'll have to get it into solution before that. You may dissolve it in >8M Urea and if still opaque put it at pH13 for a few minutes before neutralization. As you work with a pellet, you shouldn't have much problem to keep the volume and therefore dilution to a minimum. After dissolving, you can do a Bradford assay.
Usual disclaimer: All methods to determine the absolute protein concentration in solution are biased.
The pellet obtained after TCA/acetone precipitation is the extracted plant proteins, not the plant residue from initial grinding. This pellet needs to be resolubilized in a buffer containing chaotropes like urea or detergents like SDS before performing further analysis like the Bradford assay. The buffer composition will depend on downstream applications. Vortexing or sonication, and incubating at room temperature can help with resolubilization. Only after successfully resolubilizing the pellet can you proceed with further treatments like reduction/alkylation and protein digestion.
In the TCA/acetone extraction method for plant proteins, the pellet collected after centrifugation contains the extracted proteins, not the initial plant residue. To resolubilize the dried protein pellet, use a suitable buffer with a chaotropic agent (like urea or thiourea) and a reducing agent (like DTT), and apply sonication or vortexing to aid dissolution. After resolubilization, you can perform the Bradford assay to quantify protein concentration, ensuring the buffer is compatible with the assay.
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