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problem with amonium sulfate precipitation - What should i do ? (Apr/17/2004 )

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Maybe try a simple gel filtration (size) to purify your protein...


Tom




QUOTE (aparna @ Nov 23 2005, 09:28 AM)
hi!

i am working with a multidomain protein. it doesn't bind to any affinity, ion-exchange or hydrophobic column. so i tried ammonium sulfate pptn. afetr the precipitation, the protein becomes insoluble and i am not able to resuspend it in any buffer and make it soluble. this was the case even with a 10-15% ammonium sulfate.
so i shifted to zinc sulfate instead of ammonium sulfate. now i get thisprotein in soluble fraction only when i dilute it a lot. when i try to concentrate this, it precipitates again.
now when i put the protein on a gel filtration column, it comes out very close to the void volume. and plus this fraction is not pure at all.
can any body kindly suggest how to purify this protein?

thanx
aparna

-tom_vd_donk-

Ammonium Sulphate should disolve up to 4.1M , so a 4M solution in distilled water should be no problem... But normnally you use a saturated solution to mix with your protein solution, to get a wanted ammonium sulphate percentage (ie 1 volume saturated ammonium sulphate + 1 volume of your protein solution -> 50% ammonium sulphate precipitation)


greetings Tom







QUOTE (haohao @ Aug 20 2005, 01:09 PM)
Hi everyone,

I was trying to prepare 4M ammonium sulphate solution for my precipitation. I'm adding water to it but it won't dissolve completely after long hours of stirring. Can anyone help providing some details on how to prepare ammonium sulphate solution of 4M?

Thanks a lot.

haohao

-tom_vd_donk-

QUOTE (amir1979 @ Apr 17 2004, 07:44 PM)
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 . huh.gif
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

What do you know about your protein? For example, do you know the pI? Can you put your lysate onto an ion exchange column? This should enable you to get rid of lots of other proteins and concentrate your protein.

As for Aparna, have you tried ion exchange at the "wrong" pH? Remember that the calculated pI is only theoretical. Your protein may have charged patches that mean it will act counter-intuitively to what "the texts" say. You could also try mild reverse-phase chromatography.

-swanny-

QUOTE (aparna @ Nov 23 2005, 09:28 AM)
hi!

i am working with a multidomain protein. it doesn't bind to any affinity, ion-exchange or hydrophobic column. so i tried ammonium sulfate pptn. afetr the precipitation, the protein becomes insoluble and i am not able to resuspend it in any buffer and make it soluble. this was the case even with a 10-15% ammonium sulfate.
so i shifted to zinc sulfate instead of ammonium sulfate. now i get thisprotein in soluble fraction only when i dilute it a lot. when i try to concentrate this, it precipitates again.
now when i put the protein on a gel filtration column, it comes out very close to the void volume. and plus this fraction is not pure at all.
can any body kindly suggest how to purify this protein?

thanx
aparna


Have you tried acetone precipitation? Worked fine for me...:

1. Add 4 Volumes of Ice cold Acetone
2. Incubate for 30 min on ice or at -20°C
3. Centrifuge 10 min at max speed in benchtop centrifuge. Discard supernatant and air dry the pellet.
4. Optional: Wash pellet with 100 mikrol ice-cold ethanol and air-dry the pellet
5. Resuspend pellet in buffer for downstream application.


Hope it works wink.gif

-kylvalda-

kylvalda what Buffer we should use at the end, for Resuspending the final pellet for the downstream application? (by acetone precipitation as you have suggested), thanks.

Leo

-leonardu-

QUOTE (Antigen @ Apr 19 2004, 11:30 PM)
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.



Dear,

i just wanna ask you, how do you know that ammonium sulphate precipitation will work for protein with MW more that 20 kDa?
do you have the literature about this?
that would help me, because i work with enzyme that has MW 5-10 kDa.

Thanks in advance.

-vivale-

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