Molecular Techniques and Methods

Desalting and Concentration of Dilute Proteins

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

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.


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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