Hello. My name is David Vizarraga and I am a PhD student. I am trying to purify a protein but I always have a oligomer mix in the solution and I can't crystallize it (because I need a pure solution). When I did Ultracentrifugation for determine the different oligomers, It broke in the process and I obtained only 1 peak ( the linear monomer). Why is it happen? and What can I do to solve this problem? Maybe Testing a lot of buffer for that The interactions between monomers are more strong.
Thank you
I think you are saying that you have a mixture of different sized oligomers of the protein and you need to prepare a homogeneous population. You found that during (analytical?) ultracentrifugation you had only the monomer, even though you started the run with a mixture of oligomers?
My interpretation is that you have a protein that is a monomer below a certain concentration (reached during the centrifugation), but when concentrated it has a tendency to aggregate (non-specific interaction) or oligomerize (specific interaction).
I recommend exploring different conditions of salt and pH to see what effect that has on the concentration-dependent oligomerization/aggregation of the protein. You can do this most easily by dynamic light scattering.
Why do you spin you protein for 2h at 13,2 RPM? This is not an ultracentrifugation
http://www.slideshare.net/sajjadrather/sageer-ppt
Hello Adam thank you for your answer, We will test to block the exposed K residues and Then We will prove too some buffers.
Hi Paola, thank you for your answer, First We have a solution where there are many different oligomers and We wanted identify it through ultracentrifugation. But When we did it, We only obtained 1 peak ( the monomer ). So to prove that the centrifugation can broken the oligomers/aggregates to the monomer We spin the protein for 2h at 13,2 RPM.
Thank you.
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