Ammonium Sulfate precipitation...issues - (Sep/23/2006 )

Hi. I am involved in a multistep purification of a recombinant enzyme from tobacco. We first perform sequential ammonuim sulfate cuts (30% pH 4), then pellet at 70% (pH8). Dialysis o/n followed by affinity chromatography. Elution is with a LMW reversible inhibitor in PBS. Final volume is 250mL. Need to get down to 25mL to facilitate dialysis. We use 70-80% ammonium sulfate for this. Occasionally we don't get a pellet but rather a clumpy mess with an oily layer on top. Does anyone know what this is...and how to avoid it? Thanks. --Brian

hi brian

i really would not know what the messy thing is, especially since as i understand, it is coming after a step of affinity chromatography. in the crude, amm sulphate being a non-specific precipitant, also brings down things other than proteins with it, but after a chromatographic step, i don't know.

if you are doing an affinity chromatography, you are reasonably sure that only your rec enzyme will stick to the matrix and the others will flow through, so have you tried doing a desalt of the initial homogenate on something like sephadex g-25, then loading the void volume eluate onto the affinity matrix, and elute with whatever you are using. if you wish to bring down the volume of the active eluate you may then do a amm sulphate precipitation to concentrate, or you could use centrifugal concentrators from centricon.

- viv