Biomedical Engineering Reference
In-Depth Information
is much better conserved by salt precipitation than by acidic pre-
cipitation. The salt concentration needed for precipitating a protein
depends on its nature and on temperature; therefore, salting out
may be used for fractionation of protein mixtures (cf. Protocol 4.3).
Fractionation conditions are expressed as “% saturation”. Ta-
ble 8.19 and 8.20 give the amount of solid ammonium sulfate nec-
essary to get a distinct degree of saturation starting from a certain
starting saturation.
To precipitate a protein, mix its solution either at RT or 4
◦
C
with the required amount of saturated ammonium sulfate solution
(consider the different concentration of (NH
4
)
2
SO
4
at RT and 4
◦
C).
As an example, mix 6 vol.ofproteinsolutionwith4vol. of saturated
ammonium sulfate solution to get 40% saturation.
Important!
Saturatedammoniumsulfatehastohaveasolidsalt
within the flask. When preparing fresh solution, saturation equi-
librium is reached after several hours.
Allowtheprecipitatetoformovernightattherequiredtemperature
(do not store in a refrigerator if “% saturation” is made for room
temperature). Spin with 2000
g for 10 min, wash the pellet by
suspending in ammonium sulfate solution of the indicated degree of
saturation, spin again and decant the supernatant. If the precipitate
contains the wanted protein, solve it in pure water and dialyze
against an appropriate buffer.
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3.7.3 Precipitation Using Organic Substances
By addition of up to 80% (v/v) of organic solvents such as methanol,
ethanol, or acetone to protein solutions, a formation of precipitates
occurs. Varying the solvent concentration allows a fractionation as
described for salting out. Because organic solvents tend to denature
proteins at temperatures above 10
◦
C, precipitation and all further
stepshavetobeperformedat0
◦
C or below. Some buffer salts
may precipitate also at elevated concentrations of organic solvents;
therefore, the ionic strength of buffers should not be above 0.2.
After collection of the precipitate by centrifugation, the pellet
is washed and resuspended in a suitable buffer of appropriate vol-
ume. Insoluble, denatured material is removed by centrifugation
or filtration.
A very special example of protein precipitation by organic sol-
Removal of SDS
vents is the removal of SDS and other detergents from PAGE sam-
ples prior to protein determination: Mix 0.1 ml sample with 0.4 ml
methanol and centrifuge at 9000
g after 1 min. Add 0.1 - 0.2 ml
chloroform and centrifuge again. Make a two-phase system by ad-
dition of 0.3 ml ddH
2
O, centrifuge and collect the lower, organic
phase together with the interphase zone. Add 0.3 ml methanol to
the organic phase and centrifuge for 2 - 3 min. Aspirate the liquid
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