I plan to follow a protocol that we use in our lab for protein extraction, however I am afraid to lose proteins in the first steps. We start with frozen cell pellets of gram negative bacteria grown in high Fe(II) and Fe(III) concentration. The cell pellet is resuspended in buffer containing 4% SDS and 10 mM EDTA. By this the cells should be already being lysed and the proteins exposed to the remaining iron of the pellet, EDTA and SDS. Subsequently the lysate becomes boiled followed by sonication, reduction of cysteine disulfide bonds and alkylation of reduced cysteine. After centrifugation of the sample only the supernatant is used.
My question is, why is most of the protein considered to be soluble under these conditions?
During previous project I have purified specific proteins and always tried avoiding precipitation, however I worked with active/native proteins. Maybe that made me thinking that everything can precipitated your proteins. Here I am working with boiled proteins, maybe they do not react to metals, or other chemicals that would precipitate native proteins.
Unfortunately, I could not find literature helping me understanding the solubility behavior of denatured proteins.
The presence of 4% SDS will denature your proteins and impart a net negatively charged coat on the unfolded proteins. This will enhance their solibility and prevent aggregation and precipitation.
Protein masked and denatured by SDS and becomes negatively charged so completely soluble. Tsepo is right.
Thanks for your helpful answer. I knew the function of SDS only in context of SDS PAGEs, I did not know that it makes denatured proteins in general more soluble.
However, your answer is not absolute satisfying to me.
I have read that you should not use SDS PAGEs for seperation of trasmembrane proteins, because they aggregate at the pH of which the electrophoresis is operated (Running buffer pH and most isoelectric points of transmembrane proteins are not favorable.).
This means, even though SDS makes them more soluble it seems that it is only to a certain extant. When a small pH change can already cause aggregation, how can you be so sure that the SDS is enough to prevent insolobility of the proteins during the extraction process?
Thanks for the discussion.
SDS-PAGE can be used to separate transmembrane proteins. Sometimes, it is better not to heat the samples, however.
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