Biomedical Engineering Reference
In-Depth Information
TCA precipitation
To the sample containing at least 100
µ
g of protein per milliliter
add one-seventh of Soln. C to a final concentration of 10% TCA.
Incubate on ice for 30 min and centrifuge. Remove the supernatant
(save it in case the protein did not precipitate) and wash the pellet
twice with 100
µ
l ice-cold acetone each. The sample is dried in air.
Acetone precipitation
Dialyze the sample against three changes of 100 vol. of Soln. B.
Lyophilize the sample, for example, in a SpeedVac, and then add
50
µ
µ
l of Soln. D and incubate at −20
◦
C for
3 h. Centrifuge, remove supernatant and wash twice with ice-cold
acetone. The air-dried sample is subjected to further investigation.
l of ddH
2
O. Add 450
References
Braats JA, McIntire KR (1978) In: Catsimpoolas N (ed.) Electrophoresis
'78, Elsevier, North Holland, New York
Stone KL, Williams KR (1993) In: Matsudaira P (ed.) A practical guide
to protein and peptide purification for microsequencing, 2nd edn.
Academic Press, San Diego, p 48
2.5.2 Removal of SDS
Since SDS is practically not dialyzable, it must be removed by other
techniques: extraction, precipitation, electroelution, and washing
out from immobilized proteins.
Removal by extraction
A
anhydrous acetone
20
, triethylamine, acetic acid, ddH
2
O 85:5:
5:5 (v/v/v/v)
For extraction Henderson et al. gave the following procedure.
The sample is lyophilized after electroelution. To the dry sample
add 500
µ
l of Soln. A. After vortexing the mixture is incubated
in an ice bath at least for 1 h.Thenthesampleiscentrifugedin
a refrigerated centrifuge with minimal 3000
gfor10min.The
supernatant is removed and the remaining pellet (sometimes not
visible) is extracted twice with Soln. A as described. The remaining
air-dried precipitate is free from SDS.
×
Removal by electroelution
The SDS-containing solution is supplemented with an excess of
a nonionic detergent, e.g., Triton X-100. The electrode buffer is
a buffer with low ionic strength,
without
SDS, and pH nearby the
pI of the protein of interest. During electroelution SDS migrates to
the anode.
20
Anhydrous acetone is essential, since a water content above 5% de-
creases the protein yield by precipitation.
