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Proteomics Protocols

For more proteomic protocols, please see our external links to proteomic protocols.

Methods, Procedures and Techniques for Proteomics:

2-D Gel Electrophoresis Protocols

After separation, the proteins are separated out on the 2-D gel, however are invisible to the naked eye. To visualize the proteins on the gel, very sensitive staining and detection methods of the proteins must be carried out.

This is due to several facts:

  1. The gel lanes can only accept a certain volume of cell lysate or protein solution.
  2. Although the lysate/solution can be precipitated and concentrated to load more protein, usually less protein is loaded on the 2D gel as excessive protein amounts make it difficult to analyze the gel due to large spot sizes.
  3. The separation of a large amount of protein (mgs), or even thousands of different proteins from one small lane, into a relatively large two dimensionsional gel generates spots of proteins that are very low abudance / concentration (ng or picograms!) that are impossible or difficult to detect by ordinary stains or protein detection methods.
  4. A low abundance / low copy number protein can be separated, but may not even be detected due to the fact that the most sensitive stains only have a detection limit of about a 1/2 a nanogram. Picogram spot levels are currently very difficult or impossible to detect with staining.
In 2D electrophoresis, very sensitive stains are therefore required. These proteins can then be detected by a variety of means, but the most common is silver staining and coomasie stain.

Coomasie Staining Protocol

Coomassie dye binds to proteins via physisorption to amino acids such as the aromatic amino acids, arginine, and histidine. Coomasie is also used in the Bradford Method. Sensitive non-toxic coomasie stains have been generated which allow visualization of spots containing less than 1 ng of protein.

SYPRO Ruby Staining Protocol

SYPRO RUBY is a very sensitive 2D protein detection method stain, allowing visualization of about 1/2 a ng of protein at a given spot. The disadvantage is the method requires UV light to visualize the stain, thus requiring the usage of a UV light. This makes cutting of the spots difficult and potentially hazardous due to possible UV exposure.

Silver Stain Protocol

In 2-D gel silver staining, a silver colloid is applied to the gel. The silver bonds to cysteine groups within the protein. The silver is then darkened by exposure to UV light. The darkness of the silver in the spot, can be related to the amount of silver and therefore the amount of protein at a given location on the gel. This measurement can only give approximate amounts, but is adequate for most purposes.

In Gel Trypsin Digestion Protocol

What exactly is in gel trypsin digestion? Protocol and procedure for In Gel Trypsin Digestion.



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