I am actually doing protein precipitation using AS as first step of purification of an alpha-amylase. I grew my Bacillus strain an optimized medium for 24h then I spinned-down at 4500 rpm for 30min at 4°C. The supernatent was saturated at 50 and 80% AS. The pellet was redissolved in Potassium phosphate buffer 50mM and dialysed against the same buffer (200V).
The main problem is the proteins precipitate in the dialysis bag and I see at the bottom a white precipitate. Protein concentration at that point of the solution is about 2000 mg/ml.
Can this high protein concentration be the reason? if yes, what is the appropriate concentration to dissolve my proteins?
2 g/ml is way too high (i do not even think it is possible). Is the pellet soluble even if not running dialysis?
The pellet resuspend in the buffer but after some time it precipitate to the bottom of the tube
In the picture you can see the protein precipitate in a tube and even in the dialysis bag
The precipitation process most likely denature some of the protein molecule
Does this mean that the white precipitate is denatured proteins?
PS: enzyme assay gives good activity
You could have a combination of denatured and native protein. The presence of solids will contribute towards further precipitation. Are you sure you have 2 g/ml of dissolved protein?
Actually I am not sure, I suspect Bradford assay to give wrong estimation...especially that concentration increased compared to crude enzyme which is not normal
If that precipitate is protein, then there is a tremendous amount of it. I think you should lower the concentration when you do the ammonium sulfate precipitation by at least 10-fold, maybe more.
Bonjour,
Vous travaillez sur quelle type de protéines? protéines totales ou globulines?
Try with ultra filtration ampicon centrifugal unit. It is easier and hassle free to use. Moreover, protein concentration loss will be negligible.
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