Purification using Low-Salt Lysis Buffer & Downstream app. - (Aug/21/2010 )
Hi. I discovered that my GST-fusion protein can only be present in soluble fraction at NaCl concentration as low as 30mM of lysis buffer. If I would like to purify my protein using GST binding resin, will such a low-salt condition bring disadvantage to my purification? Since it only solubilize at low-salt condition, I think I only can use low-salt in washing and thrombin cleavage buffer for subsequent purification step, because it will aggregate once present in high-salt condition, right?
Usually, the salt in lysis buffer refers to NaCl. Can I substitute it with KCl, MgCl2? Or they all will just have the same effect?
The protein will be subjected to enzymatic assay. The enzymatic assay buffer contains moderate salt (~100mM). Do I have to reoptimize the enzymatic assay buffer? I am worried if the protein will instantly aggregate once I add into the assay reaction buffer!
All opinions are welcomed! Thanks!
substituting kcl or mgcl2 will cause their own problems. kcl is generally more potent than nacl in its effect on proteins, mgcl2 has other effects. stick with nacl.
lower concentrations may effect non-specific binding to the resin but may still be okay (try it).
lowering salt in the assay should have little to no effect, depending on the enzyme (some may change conformation). try it at varying salt concentrations.
mdfenko on Mon Aug 23 05:41:45 2010 said:
substituting kcl or mgcl2 will cause their own problems. kcl is generally more potent than nacl in its effect on proteins, mgcl2 has other effects. stick with nacl.
lower concentrations may effect non-specific binding to the resin but may still be okay (try it).
lowering salt in the assay should have little to no effect, depending on the enzyme (some may change conformation). try it at varying salt concentrations.
Thank you mdfenko for the reply
Since my protein become more soluble when I reduced the salt concentration in lysis buffer, does it mean my protein not being expressed as inclusion bodies in E.coli? Is there any methods to know whether the protein is actually being expressed in soluble form, inclusion bodies, or the insolubility(aggregation) is due to lysis buffer?
My lysis buffer consists of 50mM Tris-HCl (pH8), 30mM NaCl, 10% glycerol and BME. Is there any parameters I can play with? Or any chemical/additives can be added to help this condition?
Lastly, even though more soluble protein was obtained, I failed to bind it to GST binding resin. I really have no idea what to do further. I have considered to refold the protein. But our lab has never do protein refolding. We are lacking of experience, protocol and even equipment.
