3 replies to this topic

[carollee]

I have done ammonium sulfate precipitation after obtaining my crude from homogenizing with buffer (pH 6, value is above the pI of enzyme). It seems like the protein concentration in my dialyzed sample (after ammonium sulfate precipitation) is higher compared to my crude, in addition to low purification fold.

 

Is there a way where I can reduce the protein concentration after dialysis without reducing the enzyme activity? 

[mdfenko]

the protein concentration after salting out is dependent on the final volume. so your protein concentration can be higher than the crude extract.

 

did you perform a bulk or fractional ammonium sulfate precipitation?

 

a fractional precipitation can increase the specific activity of the enzyme by selectively removing contaminating proteins.

 

unless you also increase the relative concentration of an inhibitor of your enzyme.

[carollee]

the protein concentration after salting out is dependent on the final volume. so your protein concentration can be higher than the crude extract.

 

did you perform a bulk or fractional ammonium sulfate precipitation?

 

a fractional precipitation can increase the specific activity of the enzyme by selectively removing contaminating proteins.

 

unless you also increase the relative concentration of an inhibitor of your enzyme.

 

I did a bulk precipitation. My crude volume is 200mL and the final volume after precipitation is 20mL. Also, after dialysis the sample formed aggregates. Would fractional ammonium sulfate precipitation help increase specific activity and remove aggregation?

[mdfenko]

the aggregation is mostly due to protein denaturation. some will occur because the concentration is too high, some will occur just because you precipitated them, some due to handling during resuspension and changes in medium.

 

fractional precipitation will partially purify the protein of interest, especially if you actually bracket the salt concentration range in which your protein precipitates.