iam trying to purify a protein from lactobacillus which is having molecular weight of around 48.96 kDa. the theoretical pi of the protein is 9.54. Anyone please suggest me which buffer i hav to use for FPLC.
You can use Tris-HCl buffer with pH 8. Your protein will be positive in this pH and most other proteins will be negative. Then, you can pack your column with SP-Sepharose beads which is a strong cation exhchanger. Your protein can be eluted with increasing salt concentration in the buffer. If you have many contaminants, then you can use a weak cation exchanger.
Hi there,
Knowing the pI value, there is the golden rule saying that if you want to limit protein unstability you have to work at a pH value at least one unit away from pI. In your case, it means working under pH8.5 or above 10.5. At the latter pH, I don't think any protein would be happy therefore pH8.5 or below should be fine.
Knowing a little more about the protein will help before suggesting chromatography resins and operating conditions. Is this a recombinant protein? Is it procaryotic or eucaryotic? Is it over expressed and is it enzymatic? If it were a recombinant mammalian non-enzymatic protein that is highly over expressed and well folded then operating at pH 6, for example, using SP ImpRes small bead resin at greater than 30 cm bed height would like give good resolution. Your protein would have an exceptional and high positive charge that would out compete the majority of the positively charged proteins that have average pI's and at the same time out compete on the basis of mass action due to the over expression. This is one scenario but I don't know enough about your protein.
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