Molecular Techniques and Methods

Coomassie Blue Staining of Proteins in Gels

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The detection of protein bands in the gel by Coomassie Blue Staining depends on nonspecific binding of a dye, Coomassie Brilliant Blue R, to proteins. The detection limit is 0.3-1 ug/protein band.




MATERIALS AND SOLUTIONS

Fixing Solution (100 ml)
50% Methanol ------------------------------- 50 ml of 100% Methanol
10% Acetic acid ----------------------------- 10 ml of Acetic acid
Deionized, distilled H2O ---------------------- 40 ml
  • Use Fixing Solution only once.


    Coomassie Blue Staining Solution (100 ml)
    50% Methanol ------------------------------- 50 ml of 100% Methanol
    0.005% Coomassie Brilliant Blue R ---------- 500 ul of 1% Coomassie Brilliant Blue R
    10% Acetic acid ----------------------------- 10 ml of Acetic acid
    Deionized, distilled H2O --------------------- 40 ml
  • Dissolve the Coomavsie Brilliant Blue R in methanol before adding acetic acid and water.
  • Use Coomassie Blue Staining Solution only once.


    Destaining Solution (100 ml)
    5% Methanol --------------------------------- 5 ml of 100% Methanol
    7% Acetic acid ------------------------------- 7 ml of Acetic acid
    Deionized, distilled H2O ---------------------- 88 ml




    PROCEDURES

    1. Place the polyacrylamide gel(s) in a plastic box and fix the gel with Fixing Solution for 5-10 min.
    Agitate slowly on an orbital shaker.

    2. Pour out Fixing Solution.
    Stain the gel with Coomassic Blue Staining Solution for 1 hr.
    Agitate slowly.

    3. Pour out Coomassic Blue Staining Solution.
    Rinse out with distilled water briefly.

    4. Destain the gel with Destaining Solution for 1 hour to overnight.

    5. Replace with new Destaining Solution occasionally. Agitate slowly.

    6. Continue destaining until blue bands and a clear background are obtained.

    7. Store gels in 7% Acetic acid or distilled H2O.
  • Do not store gels in Fixing Solution. The protein bands will everaually disappear.

    8. To maintain a permanent gel record, the gel may be dried.
  • Place the gel between two cellophane membrane backing sheets that are slightly larger than the gel.
  • Place this sandwich between two sheets of Whatman 3MM filter paper and dry in a gel dryer at 80oC for 60 min.




    NOTES

  • Coomassie Brilliant Blue R binds nonspecifically to proteins. Since the dye does not bind to the polyacrylamide gel, proteins will be detected as blue bands surrounded by clear gel zones.




  • KIT INFORMATION




    REFERENCES


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

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