I want to determine ideal pH of my buffers to isolate my protein. The isoelectric point of my protein is 9.54 (from expasy). What must be the pH of my buffers to make the purification go smoothly?
Hi there,
In theory it's better to avoid pI region which may be destabilising for the protein by keeping working pH at 1 unit away from pI. In your case working around neutral pH should be fine (between 7 and 8). This is theory and experiments will actually tell you about what pH is the best for conservation.
ideally at least 1 unit away from the pI.
The best way to determine is to extract (into some low molarity buffer), split into several parts and exchange the buffer (or add something more concentrated. Store like this for week or two and check the activity where will be the most.
A few issues you might want to consider:
1) First you need to know the optimal pH for the physiological activity of your protein (you don't want to inactivate your protein by using buffer with pH that might cause it). If the pH for your protein activity is around physiological pH ~ 7.4 you need to try to use a buffer with this pH.
2) The general rule is: the far the pH of the buffer from the isoelectric point of your protein the better extraction and solubility of your protein are going to be. When the pH of the buffer is close to the isoelectric point of the protein the protein is the least soluble and prone to aggregation.
3) If your protein has a hydrophobic nature you need to use higher salt concentration of your buffer (and/or some detergents).
4) The composition of your buffer should also be selected based on the next step of the purification of your protein (after extraction or cell lysis).
5) I would recommend to start from something like 50 mM HEPES or Sodium Phosphate, pH 7.4. But it depends on your particular protein and the buffer optimization requires some time and experimental observations.
Based on my experience, heres what I would suggest. Since your protein has a pI above 9.5, you might want to consider strong cation exchange chromatography for pH range of say 3-8 and elution with increasing salt concentration at the respective pH. The choice of buffer would be recommended by the resin/ column manufacturer for the particular pH. Once you have established the optimum pH value, you could fine tune your purification using a weak cation exchanger.
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