Protein Precipitation



AMMONIUM SULPHATE PRECIPITATION

Ammonium sulphate precipitation is the most commonly used salt ppt because it is highly soluble in water, it stabilizes most proteins in solution (although it may denature some MAbs), and helps reduce the lipid content of the sample.

Ammonium sulphate ppt removes up to 50% of contaminating protein, and therefore reduces the load for subsequent chromatography. Most precipitated proteins retain their activity and native conformation, and can be redissolved easily. Ideal for crude and dilute samples.

At 0oC a saturated solution of ammonium sulfate is 3.9 M. This is called a 100% solution (because it is the maximum amount of ammonium sulfate you can have in a solution - not because it contains 100 g/100 ml). Generally some proteins will precipitate at 20%, others won't precipitate until close to 80%, but most proteins will precipitate at 80% or less ammonium sulfate concentration. Sometimes solid ammonium sulfate is added, but generally there is less risk of denaturing the proteins and a clearer separation of precipitated proteins if a saturated solution of ammonium sulfate is added.

To make 100% saturated ammonium sulphate at 25oC:
To 1000 ml dH2O add 766.8 g of (NH4)2SO4. pH to 6.8-7.0 with 5 or 10 M NaOH.
So, there is 541.2 g of ammonium sulphate per 1 of saturated solution.

To precipitate 2.5 ml of a solution by making it to 80% with ammonium sulfate would mean you would need to add 10 ml of saturated ammonium sulfate (SAS):
  • Chill in ice-water for 20 minutes.
  • Spin 15 minutes in cold at a minimum of 13,000g (probably better to use the high speed centrifuge at 14,000 rpm which is 23,600g).
  • Remove supe (and save if desired).
  • Resuspend pellet in relevant solvent.
Sometimes we precipitate using less than 80% solution.
A reference with information about ammonium sulfate precipitations is in the binder.

Alternatively, to fit in a microfuge tube:
  • Take 300 µl supe + 1200 µl saturated ammonium sulfate and ice 20 minutes.
  • Spin 15 minutes at maximum speed of microcentrifuge.
  • Remove supe. Resuspend pellet in relevant solvent. (Of course, if you know you can precipitate your protein with less than 80% ammonium sulfate, then you can accommodate more of the sample into the tube.)

TO MAKE A SOLUTION 20% IN AMMONIUM SULFATE (FROM 100% STOCK) ADD 0.25 VOLUMES OF 100% AMMONIUM SULFATE.

Here's an example: Say you have 3 mls of a solution and want to make it 20% ammonium sulfate. The final volume will be 3 mls + x mls where x is the amount of ammonium sulfate you need to add.

Using:
(Final V) x (final C) = (V of concentrate added) x (concentrate C).

Then:
(3 + x) x (20%) = (x) x (100%)
60 + 20x = 100x
60 = 80x
x = 3/4 or 0.75 ml.
so, to bring a 3 ml volume to 20% ammonium sulfate you will need to add 0.75 mls of 100% concentrate.

TO MAKE A SOLUTION 40% IN AMMONIUM SULFATE (FROM 100% STOCK) ADD 0.67 VOLUMES OF 100%% AMMONIUM SULFATE.
(3 + x) x (40%) = (x) x (100%)
120 + 40x = 100x
120 = 60x
x = 2 mls (or 0.67 volumes).

TO MAKE A SOLUTION 67% IN AMMONIUM SULFATE (FROM 100% STOCK) ADD 2 VOLUMES OF 100% AMMONIUM SULFATE.
(3 + x) x (67%) = (x) x (100%)
200 + 67x = 100x
200 = 33x
x = 6 mls (or 2.0 volumes).

TO MAKE A SERIES OF AMMONIUM SULFATE CUTS (USING 100% STOCK)
  1. To make the 20% cut, add 0.25 volume of 100% ammonium sulfate (3.9 M for 0oC)
  2. To convert the supernatant from that cut to 40%, you will need to add 0.42 times your original 3 ml volume (before you added ANY ammonium sulfate).
    You can solve step 2 as follows:
    where y is the additional volume of 100% ammonium sulfate you will need to add.
    (3.75 + y) (40%) = (3.75 x 20%) + (y x 100%)
    150 + 40y = 75 + 100y
    75 = 60 y
    y = 1.25 ml
    So, you will need to add an additional 1.25 ml of 100% ammonium sulfate (where 1.25 ml is 0.42x the starting volume of 3 ml) to make the sample to 40% ammonium sulfate.
  3. To convert from 40% to 67% you will need to add 2.6 times your original 3 ml volume.
    You can solve step 3 as follows:
    (5 + z) (67%) = (5 x 40%) + (z x 100%)
    335 + 67z = 80 + 100z
    255 = 33 z
    z = 7.7 ml
    So, you will need to add an additional 7.7 ml of 100% ammonium sulfate to make the sample to 67% ammonium sulfate.

NOTE: Virtually all hemolymph proteins come down by 67%.

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Last update Feb 21, 2001
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