Protein analysis

Visualization of proteins in SDS-PAGE gels

Visualization of protein bands is carried out by incubating the gel with a staining solution. The two most commonly used methods are Coomassie and silver staining. Silver staining is a more sensitive staining method than Coomassie staining, and is able to detect 2–5 ng protein per band on a gel. Many protocols are available but in order to increase reproducibility, use of a commercially available kit is recommended. Silver staining of proteins depends on the reaction of silver with sulfhydryl or carboxyl moieties in proteins and is therefore not quantitative, with some proteins being poorly stained by silver. In addition, after silver staining the protein becomes oxidized and cannot be used for further applications, such as sequencing. Coomassie staining, though less sensitive, is quantitative and Coomassie-stained proteins can be used for downstream applications.

Materials required

  • Coomassie staining solution (see table Coomassie staining solution)
  • Destaining solution (see table Destaining solution)
  • SDS polyacrylamide gel containing separated proteins (see Separation of proteins by SDS-PAGE)
Coomassie staining solution
Composition of working solution Component  Amount per 100 ml
 
0.05% (w/v) Coomassie Brilliant Blue R-250 Coomassie Brilliant Blue R-250 50 mg
40% (v/v) ethanol
Ethanol
Dissolve then add:
40 ml
10% (v/v) glacial acetic acid Glacial acetic acid 10 ml
50% (v/v) water Water 50 ml

Destaining solution
Composition of working solution Component  Amount per 100 ml
 
40% (v/v) ethanol Ethanol 40 ml
10% (v/v) glacial acetic acid Glacial acetic acid 10 ml
50% (v/v) water Water 50 ml

  1. Incubate the gel in Coomassie staining solution for between 30 min and 2 h with gentle shaking.

    Tip: Coomassie Brilliant Blue R reacts nonspecifically with proteins.

  2. Gently agitate the stained gel in destaining solution until the background becomes clear (1–2 h).

    Tip: A folded paper towel placed in the destaining bath will soak up excess stain and allow the reuse of destaining solution.

After destaining the proteins appear as blue bands against a clear gel background. Typically, bands containing 50 ng protein can be visualized.

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