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Study Notes: Visualisation of Protein Bands

To read the results of electrophoresis the bands of protein formed in the gel must be visualised in some way. Usually the gel is carefully released from the glass gel plates and then treated to fix and stain the proteins. The most common methods of visualisation are to stain the proteins or to radioactively label the proteins during their synthesis and expose the gel to a piece of X-ray film. The radiation emitted from the protein bands causes a dark band to form on the X-ray film. This process is called autoradiography or fluorography.

However, prior to staining, the proteins need to be immobilised in the gel matrix so that diffusion of the bands does not occur during handling of the gel after the electrophoresis run. Diffusion would lead to fuzzy bands instead of discrete tight bands.

Fixing proteins on electrophoresis gels
Fixation precipitates the proteins in large insoluble aggregates within the gel matrix. The purpose of fixation is to:

  • prevent diffusion and keep the protein bands sharp during the staining process
  • remove gel buffer components, especially SDS.

An ideal fixative is:

  • fast, convenient and non-hazardous
  • preserves the fine protein details in the gel.

The most commonly used fixatives are solutions of short chain alcohols and acetic acid in solution and many have the additional advantage that many of the stains used are soluble in the fixative which allows fixing and staining in one step. One drawback to these simple fixatives is that they are only moderately denaturing and may not fully fix small or highly soluble proteins.

Examples of common fixatives:

  • 45% v/v methanol, 45% v/v water and 10% v/v glacial acetic acid
  • 25% v/v isopropanol, 65% v/v water and 10% v/v glacial acetic acid
  • 12% w/v trichloroacetic acid (TCA) in water (strong fixative for difficult proteins).

The fixation process usually takes one hour at room temperature but for difficult proteins TCA fixation would be extended up to three hours.

Staining proteins in gels
Chemical stains work by having a much greater affinity for the protein than for the gel matrix. They are non-specific and will stain all proteins, and laboratory coats and fingers if you are not careful!

The important characteristics of a good stain are:

  • low background (staining of the gel)
  • high sensitivity (will stain even small amounts of protein strongly)
  • large linear range (intensity of the stain is directly related to the amount of protein present)
  • ease of use.

Some commonly used stains are shown below.

Stain
Time Required
Sensitivity
Amido Black
12 hours
500 ng
Coomassie Blue R-250
16 hours
50 ng
Coomassie Blue G-250
1 - 2 hours
200 ng
Silver
2 hours
1 ng
Immunological
4 - 6hours
<10 pg

The first three are classical stains. Silver uses the same chemistry as in black and white photography. Immunology stains are used during the process commonly called western blotting (to be covered in more detail in Step 7).

Staining protocol for Coomassie Blue Stains:

  • fix gel in 50% methanol/10% acetic acid/40% water for 30 minutes to overnight
  • stain in above solution with 0.25% w/v Coomassie Blue for 2 - 4 hours
  • destain in 5% methanol/7.5% acetic acid/87.5% water for 1 - 2 hours
  • store gel in 7% acetic acid/93% water.

Drying of the gel
Fixed and stained gels may be kept indefinitely in an acetic acid solution but often a more permanent and usable form is required. Gels may be dried onto a piece of appropriate chromatography paper.

Commercially available drying units are used that keep the gel flat and remove water over about 1 - 2 hours under vacuum and with an increased temperature.

The dried gel can then be filed for future use. Note that the gel must be kept flat under some weight, as dried gels tend to curl up and will then crack if unfolded.

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