My confusion is that when we stock of aggregation prone peptide is prepared in either in denaturant like urea or in acid (TFA), is diluted to water or PBS buffer (pH 7), then sometimes I see turbidity. So that is a result of fast aggregation or precipitation.
You could have protein aggregation without precipitation. Aggregation is commonly used to describe the formation of multimeric associations, normally as intermediates in the process of forming insoluble precipitates. In the initial stages those aggregates might be soluble or partly soluble. Only afterwards, when the aggregates are larger, they become insoluble and precipitate (think of the proposed mechanism for amyloid plaque formation).
You could have protein aggregation without precipitation. Aggregation is commonly used to describe the formation of multimeric associations, normally as intermediates in the process of forming insoluble precipitates. In the initial stages those aggregates might be soluble or partly soluble. Only afterwards, when the aggregates are larger, they become insoluble and precipitate (think of the proposed mechanism for amyloid plaque formation).
Hi!
If you keep your protein solution on ice or in higher pH than what its soluble in, the proteins will sediment sown. this is called precipitation.
Where as, even after being soluble in a solution if monomeric proteins clump together to form dimeric or multimeric conformation then its called aggregation. sometimes, aggregated proteins do sediment because of their higher molecular weights than usual.
But in my case i am seeing turbidity immediately after addition of peptide to solution and on centrifugation no peptide in supernatant, when analysed by HPLC. So i just want to clarify if this can be precipitation or it can also be due to fast peptide aggregation.
aggregation is the process of peptide molecules forming higher order structures. Precipitation is the process of peptide molecules coming out of the solution; one is a description of the molecular associative state, the other is a physical process related with the solubility of the aggregates. Do you have your peptide in a stock solution in a different solvent? it might just be that your peptide isn't soluble in the conditions you are using for your experiment.
Yes my peptide is in either GuHCl or DMSO, since it is not direcltly soluble in PBS buffer or water.
Then I'd say your peptide is just not soluble. You never even have a significant concentration of the stuff in solution to describe it as aggregating.
Eugenios: I'm having similar issues. I'm not sure about if my protein form aggregates or precipitate. When I'm doing the dialysis or the concentration steps (either PBS or phosphate buffers), then I can see my sample not clear. I don't see crystals, just turbidity. Is that means that my sample is soluble or partial soluble and it is forming aggregates?
Claudia, if there is no precipitate, then the turbidity is due to aggregated proteins. An adjustment in pH may help get these in solution. Other components such as salt concentration can also influence aggregation. Turbidometry is actually a very useful type of spectroscopy used to measure formation of protein complexes or protein aggregation. If you are dealing with a membrane protein, you may need to add other chemicals (e.g. non-ionic detergents) to keep the proteins from aggregating.
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