Proteins in the final stages of chromatographic purification are
often recovered at low concentrations and require stabilization
in viscous buffers containing glycerol. Such purified protein
preparations are often concentrated by trichloroacetic acid (TCA)
precipitation to resolve the proteins by SDS-PAGE either in small amounts for analysis or in larger amounts for
obtaining amino acid sequence information. However, dilute proteins
in viscous solution may be difficult to concentrate by conventional
TCA precipitation. In this protocol, both TCA precipitation and
ultracentrifugation are used to concentrate the non-abundant proteins
in viscous solution.
MATERIALS AND SOLUTIONS
Buffer A (10 ml)
10 mM HEPES-KOH (pH 7.9) ---------------------- 0.1 ml of 100
mM HEPES-KOH Buffer
0.5 mM EDTA --------------------------------------- 10 ul of 0.5 M EDTA
1 mM DTT ------------------------------------------- 10 ul of
1 M DTT
10% Glycerol ----------------------------------------- 2 ml of
50% Glycerol
1 M KCl --------------------------------------------- 745 mg
Add distilled H2O to make a final volume of --------- 10 ml
100 mM HEPES-KOH Buffer (100 ml)
100 mM HEPES (potassium salt) ---------------------- 2.8 g
Add distilled H2O to make a final volume of ----------100 ml
Adjust pH to 7.9 with 0.1 M KOH.
Buffer B (10 ml)
125 mM Tris-HCl (pH 6.8) --------------------------- 1.25 ml of
1 M Tris-HCl
0.1% SDS -------------------------------------------- 0.1 ml of
10% SDS
100 mM DTT ----------------------------------------- 1 ml of 1 M DTT
0.004% Bromophenol blue ---------------------------- 8 ul of 5% BPB
Distilled H2O ------------------------------------------ 7.642 ml
PROCEDURES
Desalting Proteins
1. Desalt proteins by injecting small sample volumes (< 2 ml)
with an HPLC syringe or to pump large volumes of salt solutions
containing small quantities of protein directly onto a reversed-phase HPLC column by using an auxiliary pump.
The salt concentration should be kept at 0.1 M when using large
volumes by diluting the solution with water if the initial concentration
is > 0.1 M. The salt is rapidly eluted from the column, and the
protein is retained. The protein can be eluted with 50% methanol
in water (v/v) and collected manually into a polypropylene tube.
The protein may also be desalted by dialysis or precipitation.
Concentration of Dilute Proteins
2. Suspend Proteins at concentrations less than 10 ug/ml in 1
ml of Buffer A.
3. Add 135 ul of 100% TCA.
4. Incubate tube for 1 hour at 4oC.
5. Ultracentrifuge in a swing-bucket rotor at 187,000g for 1-16
h at 4oC to precipitate the proteins. Use thick-wall polyallomer tubes.
6. Wash the protein pellet with 75% acetone at -20oC.
7. Air dry protein pellets briefly.
8. Resuspend protein pellet in 100 ul of Buffer B by incubating
for 20 min at 60oC and then for 5 min at 100oC.
NOTES
KIT INFORMATION
REFERENCES
Bosserhoff A, Wallach J, Frank R (1989) Micropreparative separation
of peptides derived from sodium dodecyl sulfate-solubilized proteins.
J. Chromatogr. 473: 71-77.
Mahuran D, Clernents P, Carrelia M, Strasberg PM (1983) A high
recovery method for concentrating microgram quantities of protein
from large volumes of solution. Anal. Biochem. 129: 513-516.