Sample Preparation

• When a detergent such as sodium dodecylsulfonate (SDS) is added to a protein or nucleic acid, the detergent will associate with and unfold (denatured) proteins and nucleic acid. Denaturation of proteins makes it easier to determine their molecular weight in electrophoresis. The amount of sample required is typically 100 to 500 nanograms per sample band for proteins and 5 to 100 ng per band for nucleic acids in a total volume of about 25 to 40 microliters.
  
  

Gels

• A gel, typically made of polyacrylamide or agarose, can be prepared or purchased to separate these proteins. The gel is a lot like gelatin. It is mostly water, but is solid enough for handling. The gels contain buffer to control the pH. The gel is very thin (1 to 2 mm) and rectangular. One side looks like a comb with a lot of missing teeth. The gaps are called wells.
  
  

Sample Loading

• One places the gel in a chamber with buffer and denatured proteins are added to the wells. Samples of known molecular weights are added to the outside wells. Gels are made with varying amounts of polyacrylamide. A low gel strength (4 percent) is better for separating high molecular weight molecules, while a higher gel strength (12 percent) is used for higher molecular weight molecules. A gradient gel varies in gel strength and can separate a wide range of proteins with the loss of some resolution.
  
  

Electromigration

• With the application of a high electrical voltage, the denatured proteins move toward the bottom of the well. The higher the weight of the protein, the slower it moves. When the electricity is turned off, the proteins stop moving. One removes the gel from the chamber and rocks it in a dish with dye. Organic dyes such as Coomassie blue or metal dyes are used to stain proteins while fluorescent dyes such as ethidium bromide are used for nucleic acids.
  
  

Analysis of Results

• With the removal of excess stain, one can see that mixtures separate into bands that look like ladders. The position of the bands is compared to the distance moved by the standards to determine the molecular weight of the sample in each band. The gel can be dehydrated with alcohol and dried so it can be saved. The color intensity of the band then can be measured to determine the concentration of the protein in each band.
  
  

Protocol Development

• Standard protocols are available for working with well-known proteins. If a researcher is working with an unfamiliar sample, the gel strength, buffer type, buffer pH, voltage, run time, and stain may all need to be optimized to obtain the best separation and signal.
  
  

Protocols