Problems with inconsitant protein recovery using ammonium sulphate (AS) precipit - protein precipitation/concentration (Jan/17/2008 )
Hello, I am new to the forum and am really confused with this problem. Please bear with me, I feel that some back ground is needed, so please read and see what you think.
I am working on an extracellular protein excreted by the filamentous fungus Beauveria bassiana which has an apparnt Mw of about 35 Kda. I have chossen AS precipitation as my method of concentration becuase I am working with fairly large volumes (0.7-1 L). I am closely following a very standard protocol and have found the protein precipitates at 80% saturation.
My problem is how inconsistant the AS is precipitating the protein. Some of the time the filtrate becomes very cloudy when saturations of >60% are reached and others it remains very clear, reflecting that the proteins are not precipitating and as a result are not recovered by centrifuge. Some protein is recovered, however protein assays show that protein recovery is about 10-20x (10 mg protein vs 0.5-1 mg) greater when the filtrate become cloudy when the appropriate saturation is reached
My assumption for this inconsitancy is the protein concentration in the filtrate is greatly varied in the filtrate between batches prior to saturation. However, precautions are taken to limit proteolysis (manipulations are done at 4 C) and the cultures are grown for approximately the same time with in 4 hrs and the banding patterns using SDS-PAGE between the batches is similar especially with respect to my protein, however band intensities are differant due to protein concentration.
The main problem with all this is the activity associated with the protein is very difficult to assay for and fairly high concentrations are required so this low recovery rate is problematic.
thanks alot everyone ,
Protein conc, pH, temperature, the rate of addition of solid (NH4)2SO4 are the major variables that can affect the outcome of precipitation. Either your sample or the way you added solid are not consistant between runs.
Protein conc of filtrate is 20-40 ug/ml always, pH is consistant in filtrate, temp is held at 4C during precipitation, and the ammonium sulfate is ground into a fine powder and slowly added over 30 min always to ensure it is being rapidly dissolved. I agree with you those are the major variables, but the confusing thing is they have been considered and still my recovery is inconsistant.
Thanks for your input,