Biomedical Engineering Reference
In-Depth Information
Amersham Biosiences Application note: HiTrap chelating.
http://www.amershambiosciences.com
Qiagen TAGZyme
TM
Handbook for exoproteolytic cleavage of N-terminal
His tags. http://www.qiagen.com
3.7 Concentration of Diluted Protein Solutions
3.7.1 Acidic Precipitation
µ
/
Proteins in aqueous samples with concentrations of at least 5
ml
are precipitated by acids as trichloroacetic acid (TCA) or sulfosali-
cylic acid. The final concentration of acid should be 7.5 - 10% (w/v).
Biologic activity is mostly and antigenicity is sometimes destroyed
by acidic precipitation.
g
A
100% TCA (w/v) in ddH
2
O
Solutions/Reagents
B 10% TCA (w/v) in ddH
2
O
C 0.15% sodium deoxycholate (NaDOC) (w/v) in ddH
2
O
Cool the sample in an ice bath and add 1/10 volume of Soln. A.
Vortex and allow to precipitate in an ice bath for 30 min.Spinfor
5 min at 5000 to 10 000
g in a refrigerated centrifuge. Remove
the supernatant by aspiration with a pipet. Wash the pellet once
with 0.5 sample volume ice-cold 10% TCA (w/v) and once with 0.5
sample volume ice-cold ethanol-ether 1:1 (v/v) to remove traces of
TCA. Dry the pellet at the air and dissolve in 0.1 N NaOH or 0.1 M
Tris pH 8.0.
Using NaDOC, the protein concentration precipitable by TCA
is below 1
×
µ
/
ml. The precipitation protocol is modified as follows:
Add 0.1 ml Soln. C per milliliter to a sample. Mix and incubate at
RT for 10 min.Add0.1ml of Soln. A, cool to 0
◦
C and continue as
described above. In the presence of SDS the NaDOC-TCA precipi-
tation does not work.
Proteins from tissue homogenates are precipitated by tungstic
acid. Mix homogenates, which contain about 1% protein, with 1/10
of volume of 10% sodium tungstate (Na
2
WO
4
·
·
g
H
2
O) (w/v), and
acidify with the same volume of 0.67 N sulfuric acid. Collect the
precipitate after 10 min at RT by centrifugation. Nucleic acids are
precipitated by tungstic acid to a minor extent.
References
Bollag DM, Edelstein SJ (1991) Protein methods. Wiley-Liss, New York
3.7.2 Salting Out
Proteins are salted out (precipitated) by ammonium or sodium sul-
fate (see Hofemeister series, Sect. 3.2). The structure of proteins
