Structural Biochemistry, Proteins, Purification, Micro-Purification , Edman Sequencing

Basis for the Understanding of Micro-purification: Edman Sequencing

Edman sequencing is the ability to determine the sequence of a protein. The contribution of studying amino acids was considered an extreme advance in the studies of Structural Biochemistry. Especially in the understanding of protein structure and its function relationships, Edman sequencing has aided experiments such as the design of PCR primers, the production of synthetic peptide antigens and the creation of DNA probes. The ability to identify a particular protein can be determined through the use of Edman sequencing coupled with the use of a computer search database. Sequence analysis is upheld as the first place of importance for structural function.

The only problem presented through the production of micro-sequencing is the success of purification of proteins itself, not the technique of micro-sequencing. There is no instrument advanced enough to obtain useful information from impure amount of proteins. The only proteins that researchers are most interested in are found in low doses of abundance. That is why the preparation of a sample protein is very challenging and risky when preparing for a micro-purification process. Purifying, storing and recovering of proteins are presented as side-effects to the study of characterization of structural biochemistry. The low dose of abundance proteins also present itself as a side-effect to biological assay.

Techniques for Edman Sequencing

SDS-Page

The technique of SDS-page for the strategy of micro-purification is considered the most common and popular technique of choice in regards to physical and structural strategies. When dealing with such a low amount of abundance proteins, any multiple-step technique will lose much of the limited sample protein along the way. SDS-page is a one step technique that has no loss of a purified sample protein when it is running under gel electrophoresis. Even partially purified proteins can be further studied through electrophoresis. A partially purified sample protein can be placed onto a small-scale gel and be purified, producing a few micrograms of purified protein. Attention needs to be paid when working with small amounts of protein, especially micrograms, because a large amount of free amino termini can be lost due to alkylation from a reactive species. To avoid this issue, only the most pure of proteins can be used. The use of high quality gradient gel is the best way to preserve an understandable yield for lower amounts of protein.

2D-Electrophoresis

The use of 2D-Electrophoresis is another useful tool in purifying and separating proteins in a gel. The separation of the proteins can take place without having to do any preliminary purification. When the technique of SDS-Page fails to produce the purification of a particular protein, 2D-Electrophoresis is the next method to turn to. However, similar to SDS-Page, 2D-Electrophoresis presents some side-effects. One side-effect is that multiple 2D-electrophoresis gels are difficult to compare because the gels themselves are hard to reproduce between lab experiments. Another side-effect is that the capacity of the gel paper is poor in quality and needs to be run several times in order to collect enough protein for further analysis. Nonetheless, with technology constantly advancing in mass spectrometry and micro-purification, these side-effects will soon be eliminated.

Reference: University of California, Davis. http://msf.ucdavis.edu/micro_strat_edman.html. Last accessed: 9 Dec 2011.

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