I Have Recently I have begun a project on expression and purification of a cytoplasmic protein. When running analytical Superdex 200 the protein elutes in the void volume despite the monomer being around 45kDa.
As such I have been trying several conditions to alleviate the problem (addition of salts/detergents/and other additives). However performing lysis and subsequent SEC takes too long.
As such I am wondering if Thermal Shift/Differential Scanning Fluorimetry be used on soluble aggregates? Or would the data be too difficult to interpret due to complex misfolding pattern o, maybe the increase in melting temp be indicative of a condition that favours the aggregated state.
Any help on the topic would be greatly appreciated.
Which is the purpose of using Thermal Shift or Differential Scanning Fluorimetry?
In any case, if your protein is forming oligomers (if ordered) or aggregates (if amorphous) in an unexpected way, then, your protein is not in a good shape. Then, performing downstream assays might make little sense because you will not believe the results, no matter they are fine or ugly.
However, you might look into those assemblies to find out whether your protein is self-organizing into oligomers and that is its natural native state.
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