Researchers need to understand the expression of proteins. They also would like to understand protein expression in healthy and diseased cells.
Hence the study of proteomics, which characterises and analyses of the function of proteins in the body is crucial.
In general, a mixture of proteins is separated by HPLC the resultant major peaks are collected in a fraction collector. This collected material is then suitable for characterisation by amino acid analysis, sequence analysis, capillary electrophoresis or SDS-PAGE gel electrophoresis.
The same analytical HPLC method may be used to elute a given protein mixture. If larger quantities of the separated proteins are required, the user can very easily scale up the analytical method to semi-preparative scale. The key parameter for transferring an HPLC separation from the analytical to preparative scale is column volume. Both mass load and volume load need to be scaled in proportion to column volume. Flow rates should be scaled up in proportion to column volumes.
The HPLC method may for example, involve the use of ion exchange , gel filtration or affinity chromatography techniques, depending on the proteins of interest A uv/visible HPLC detector is often used.
If there is a limited quantity of sample, reverse phase narrow bore HPLC column (0.21 x 25cm) is useful. Analytical HPLC reverse phase columns, 0.46 x 25cm, are useful for larger sample quantities up to 500g. Semi-preparative HPLC , to 3mg/run, may be run on wider bore columns such as 2.2 x 25cm.
In some cases, it may be necessary to remove interfering and abundant proteins, such as albumin, IgG, IgA, transferrin, haptoglobin, and antitrypsin and dye terminators from human serum sample matrices. This may be performed by the use of Multiple Affinity Removal Systems or by the use of Spin columns. These systems and columns are used to pre-treat samples, before they are injected onto an HPLC column.
Samples should not be suspended in detergent or non volatile buffer containing solutions. Some detergents may bind to reverse phase columns and modify them irreversibly. Samples should not be dissolved in organic solvents, as this may inhibit the proteins from adsorbing to the stationary phase.
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