I am doing affinity chromatography for Aprotinin, I am using trypsin affinity column,equilibration and binding of aprotinin ll be done with Tris HCl buffer pH 8.0 and sodium chloride concentration of 0.3M,I just want to know the role of sodium chloride in this affinity chromatography.
why sodium chloride is used at such higher molar concentration,for binding?
if you know any better binding buffer please let me know because i am getting lower binding.
I would guess that the high salt concentration is to reduce non-specific binding of other proteins to the column through ionic interactions.
If you think that the binding is weak, you can try to lower the salt concentration to 0.2 M or even 0.1 M. SP column can do as well.
Adam is right about high salt reducing non-specific interactions with the column.
Also, aprotinin interacts with the trypsin active site using a Lysine side-chain, so high concentrations of NaCl could disrupt binding of aprotinin to trypsin. Are you sure the protocol called for 0.3 M NaCL? You could try 0.03 M NaCl.
I would doubt that binding of aprotinin to immobilized trypsin is low due to 0.3 M NaCl in your binding buffer. There can be several reasons for low yields in protease affinity chromatography. First, it could be that you immobilized much less protease than you thought (did you check the immobilization efficiency?). Next, on the first (and sometimes second) use of a new affinity column it is normal to see some protein leakage from the column. In the case you are determining your aprotinin content based on protease inhibition, you can get low inhibitory activity because a part of aprotinin is complexed to trypsin that was co-eluted from the column. Last but not least, it could be that your elution buffer represents a too mild environment to disrupt the very strong binding of aprotinin to trypsin.
Although this was not really your question, I would consider using ion-exchange chromatography as an alternative to affinity chromatography. BPTI is such a basic protein that it is very easy to purify it by IEC.
I agree with Marko in trying to change to cation exchange. We have faced troubles in the past attempting purify a basic trypsin inhibitor by trypsin-affinity chromatography. The peak eluted nicely, single and apparently pure, but the inhibitory activity decayed. Further analysis by RP-HPLC showed several peaks. Since it is a quite basic peptide, it is conceivable that free immobilized-trypsin can hydrolyze inhibitor bound to other trypsin in the vicinity. That is, binding between trypsin and the inhibitor occurs through the inhibitor reactive site, thus the remaining basic residues in the inhibitor are exposed to hydrolysis by nearby trypsin.
I also agree with the colleagues that ionic strength in the binding buffer (0.3 M NaCl) is maybe too high. If you look in literature, you could find that inhibitor elution from trypsin affinity chromatography is often accomplished by increasing NaCl or KCl concentration.
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