I am working with a DNA binding protein 180 Kda. after Heparin Purification or Gel filtration, when i used to centrifuge the purified protein at 10,000 xg, 10 mint, pellet is observed sometimes and after pellet formation, the concentration of purified protein is reduced significantly. Centrifugation is important for homogenizing for downstream assays. So how could i determine that which centrifugation speed is suitable to homogenize this protein??
The fact that the protein pelleted during a low speed centrifugation tells you that it has most likely aggregated and precipitated because it is unstable at that concentration and under those storage conditions. Additional work will be needed to prepare and store the protein in a stable, non-aggregated state.
If it was not aggregated to begin with, but the aggregation happened subsequent to purification, you could try changing the storage buffer (pH, salt), adding a stabilizer (glycerol, trehalose), and reducing the concentration.
You are having a solubility problem. DNA binding protein means it binds the phosphodiester backbone of DNA in some way by electrostatic contacts so add 500+ mM of sodium chloride to your buffer and you should be fine. But once protein has precipitated usually you have to try again from protein expression and purification.
Heparin purification is probably removing endogenously bound DNA and size exclusion is probably separating the state of the protein not bound to DNA so it is unstable.
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