I am trying to identify some small molecules in a bacterial supernatant extract. I would like to remove proteins to make mass spectrometry easier. I have tried protein crash with ACN but the concentrated supernatant extract is more soluble in water and it does not solublize in high concentrations of solvent. Would heating supernatant for 5 minutes at 100 C precipitate some proteins? My active component in the supernatant is heat resistant (at least to 100 C).
The answer is almost certainly yes, this amount of heat will precipitate some globular heat sensitive proteins. The amount will depend on durtion of heating, pH, protein concentration etc. However, it is not clear that this will get rid of all proteins you want to remove.
An easy way to separate small molecules in a supernatant extract (I am assuming cells are lysed) is to use dialysis into a suitable buffer or even water. The same can be accomplished using gel filtration. Another approach is to add ammonium sulphate salt (slowly with stirring) to about 70% saturation. This will cause precipitation of most proteins which can be removed by centrifugation. Yet anther approach is to use a mini ion exchange column (anion or cation) which will bind most proteins at a suitably high (or low) pH. There are also options with solvent precipitation etc...
Thanks Gary. I have tried this a few times now with some biologically active extracts and there looks as if there is some precipitation without too much loss of activity. I have not had the opportunity to look at it on the mass spectrometer yet
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