problem with amonium sulfate precipitation - What should i do ? (Apr/17/2004 )
dear all
I have produced alpha amylase enzyme with Bacillus licheniformis
in a volume of 2 liters . Assay on alpha amylase activity has been done and i am sure that it has amylase activity.
Now i want to concentrate my enzyme with Amonium sulfate precipitation and compare it with ultra filtration ,but in three assays that have been done till now i have nt aquire any precipitate in 85% of saturation and also any pellete .
as i persume i should have at least 1 mililiter precipitant out of my 10-2o ml of my sample but unfortunately i dont have any precipitant . 
i am very nervouse cause i should finish my practical work up to two month later . what should i do for this problem . what do you suggest ?
thanks in advance
amir golmohammadi
Iran
Amir
You are not getting any precipitate with ammonium sulfate because the overall protein concentration in the culture supernatant is low. I suggest you first bring down the effective volume of your culture supernatant. This can be done in a small batch of 100 ml. Remove 100 ml of the amylase containing supernatant and dialyze against 1 L 25mM Tris buffer containing 10 mM MgCl2 and glycerol at 50% v/v at 4C for 10 h. This will bring down the volume of your filtrate from 100 ml to 34 ml. Subject this dialysate to precipitation with ammonium sulfate at 4C and the process must be carried out for a total time of 48h. You will see a precipitate- it will be light and flaky but the active enzyme will be present in the pellet because amylases are quite stable.
Good luck and God Bless
Amir
Also- you cannot use filtration to bring your enzyme volume down because media salts will increase in concentration and most probably kill the activity of the amylase before you can use diluents. Dialysis is the preferred option.
Good Luck again and let us know if you need further assistance. Write a private note to dstf@doctor.com and one of our staff enzymologists will stay with you via email 24 hours a day until you complete your practical work. Don't worry- you are in the right place and all the right experts have read your note and understood your problem.
Amir,
Ammonium sulfate precipitation works well for relatively concentrated protein solutions only. If you have less than 0.1 mg/ml - it will be difficult to concentrate, try up to 90% and overnight in cold-room. Also this kind of precipitation works better with hydrophobic proteins and with MW more than 20kDa. I would try cold aceton or ethanol.
For best precipitation pH must be lower than protein pI, cause sulphat ion interact with positive charged groups, making protein more compact.
If it is still important for you 
Hi all,
Regarding am.sulfate ppt., I had the impression that at 4M concentration, ALL proteins precipitate...
Regarding am.sulfate ppt., I had the impression that at 4M concentration, ALL proteins precipitate...
That is true provided the protein is occupying salt 'space'. A low concentration protein can coexist with that concentration of ammonium sulfate because their individual solvations have not been interfered with. A very good point though and is used in Biochemical engineering for seeding fermentation products by adding denatured papain.
I have a different problem with AS precipitation. When I add water not all the protein resuspends after the A.S. What am I doing wrong?
The volume of water I add is the same as the starting volume of the protein sample (2ml) prior to adding the A.S. It doesn't resuspend everything and I am still left with a white precipitate. Any hints? Should I be adding a buffer and not water?
The starting concentration of the protein solution is 5mg/ml and I perform the precipitation by slowly adding A.S to the protein solution which is mixing at the time. I then leave it stirring at room temp for 10 minutes before centrifugation at 15,000g for 10 minutes at 4oC. I resuspend at room temperature. The A.S solution I made was done by adding A.S to water (boiling) until no more went into solution. I cooled it down to 4oC and spun out the remaining undisolved A.S. Is that the right way of doing that? I am assuming that this is a 100% solution.
Proteinjunkie
I think you are making a big mistake! You should add SOLID ammonium sulfate at whatever concentration you need it and then add a little bit of water ONLY if you see any undisolved ammonium sulfate at the bottom of the beaker.
Remember to add it slowly and give it a good 2 hours to mix with your protein solution.
What if I am working with only 2mls....? Same advice?