Liquid chromatography has been used for isolating proteins, peptides, and other molecules from complex mixtures. Usually a protein purification protocol contains one or more chromatographic steps. The basic procedure in chromatography is to flow the solution containing the protein through a column packed with various materials. Different proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the protein from the column. Usually proteins are detected as they are coming off the column by their absorbance at 280 nm. Many different chromatographic methods exist:
Separation of Proteins Using Molecular Exclusion Chromatography This method is basically used to separate macromolecules on the basis of number properties, such as size, charge or affinity for other molecules. Dialysis is the simplest method used for separating small molecules from proteins through a semi-permeable membrane. Pores in the membrane allow molecules up to 10KDa to pass through, but larger molecules are retained. Small molecules therefore move out into the buffer, the buffer is placed in the membrane.
Gel Filtration is also used to separate substances according to molecular size; the method involves a column filled with very small beads of an insoluble but hydrated carbohydrate polymer, which is suspended in buffer. When a mixture of molecules are applied to the top of the column, followed by buffer solution, the molecules move down the column at different rates, depending on there sizes they can be collected as they emerge from the bottom of the column, small molecules are able to enter the beads of the gel.
Ion Exchange Chromatography
Proteins can be separated on the basis of their net charge by ion- exchange chromatography; a protein with a net positive charge will normally bind to a solid phase containing negatively charge carboxyl ate groups. On the other hand if the solid phase contains positively charged tertiary amino groups, then negatively charged proteins will bind but proteins with a positive charge will not. Chemically modified cellulose is often used as solid phases for protein separation.
In conclusion, the whole process of protein isolation and purification involves 5 main steps. Firstly, the protein has to be extracted from cells, by breaking up cells (homogenisation) and secondly extraction from other cell components (centrifugation). Extraction from other cellular proteins (ultracentrifugation). Centrifugation separates components based on their densities, thirdly, precipitation steps are (soluble proteins form precipitate in solution) to recover protein from extract mixture. fourthly, use of chromatography to further separate the target protein from bulk protein in order to check the purity and further purifying by use of electrophoresis, assay procedures (based on enzymatic activity or another easily montiorable property specific to the protein. Therefore in the healthcare area where associated infections are needed to be discovered, the isolation of proteins is important, to detect and treat these infections.
There fore in a fight against Healthcare associated infection, should be made more aware and took more seriously, as it is very important for patients to stay safe when seeking treatment from healthcare practises. Strict rules in hygiene should be followed when staying or receiving treatment and the public should be aware of the treatment and consequences and what they are going to receive. Overall the detection procedures used are very good and can separated proteins so that they can be examined and be identified which protein it is.
References:
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3. Clive Dennison.. A Guide to Protein Isolation. Science,2003.
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