1. PREPARED BY: NOR HELYA IMAN KAMALUDIN helya@unimap.edu.my PTT 202: ORGANIC CHEMISTRY FOR BIOTECHNOLOGY

2. Introduction of Proteins Proteins are made up of about 22 amino acids, which are linked by the peptide bond. Methods for the quantitation of proteins are either suitable for all proteins or designed to measure individual proteins. Such specific methods may depend on either a preparative stage in the analysis or the use of a specific characteristic of the protein.

3. Separation of Proteins Separation of proteins PrecipitationElectrophoresis Immunological methods Immunoaffinity purification Chromatographic methods

4. Precipitation High concentrations of a variety of salts including sulphates, sulphites and phosphates can be used to precipitate proteins But, each fraction produced still consists of a mixture of protein Usually requires further purification. Excessive denaturation of the protein is avoided by the use of low temperatures. Salt fractionation techniques prepare protein fractions by successively increased concentrations of the salt. Various alcohols may also be used and the classical Cohn fractions of serum proteins are separated using specific concentrations of ethanol under carefully controlled conditions of temperature and pH.

5. Electrophoresis Electrophoresis has a major application in the separation of proteins because charge and molecular size are important to both electrophoretic separation and to protein structure. Conventional techniques: Use a flat supporting medium, e.g. cellulose acetate strip or open gel plate Capillary techniques: Instrumental technique and is very dependent upon the availability of commercially produced equipment and reagents. In both techniques, the key factors in the separation of proteins are the choice of the operating pH and the electrophoretic conditions to be used. The technical decisions are largely made by the manufacturer rather than analyst. It is essential, however, that the analyst can identify the molecular features of the analytical problem and then select an appropriate technique.

6. Technical aspects of electrophoresis Factors affecting electrophoresis Supporting medium Iso-electric focusing technique pH of buffer Concentrati on of buffer The methods of capillary electrophore sis performed

7. 1. Supporting medium Filter paper Has significant disadvantages, the most serious being the adsorption of proteins. Modified cellulose media such as cellulose acetate shows significantly less adsorptive effects. But, together with a uniform pore structure, it result in a greatly improved resolution. Starch and polyacrylamide gels Show improved resolution due to a molecular sieving effect. Patterns of separation for different media It is important to realize that the use of these different media for the same sample will result in separation patterns that cannot be easily compared with one another. The separation of serum protein on cellulose acetate will result in 5-7 bands, while the use of polyacrylamide gel will give 17 bands.

8. 2. Iso-electric focusing technique Iso-electric focusing technique Probably give the best resolution and many of the resulting bands are due to specific proteins. They are used mainly as a qualitative or a semi- qualitative technique due to: The large number of bands that develop Particular value when successive samples from the same source need to be compared for the presence or absence of a particular protein or for investigation of physical properties.

9. 3. Buffer pH pH of the buffer It may affects the charge carried by the protein. In theory any pH may be used, but, in practice pH values greater than the iso-electric pH of the protein give better separation than others pH values. In selecting the conditions for the separation of a particular protein mixture, a buffer pH that gives the greatest difference in the charge carried by each individual protein results in the greatest difference in velocities and hence in the final distances moved. In practice buffer of pH 8.6 are the most frequently used.

10. 4. Buffer concentration Concentration of buffer Could also affect the mobility of protein. At high concentration, the zeta potential of the protein is reduced resulting in a shorter distance of migration. However, because higher concentrations of buffer give improved resolution, a compromise concentration has to be found and buffer with ionic strength (μ) varying from 0.025 to 0.075 are frequently used.

11. 5. The methods of capillary electophoresis performed Capillary electrophoresis The various types of capillary electrophoresis are performed either in free solution or in gels. The choice of method depends on the nature of the sample and the analytical objectives. However, capillary electrophoresis including iso- electric focusing and SDS electrophoresis is particularly useful for protein applications.

12. Quantitative aspects for electrophoresis Quantitative aspects Semi- quantitative method Alternative quantitative method

13. 1. Semi-quantitative method Conventional electrophoresis In conventional electrophoresis, semi-quantitative method refer to the amount of dye bound in the pores of the supporting medium which offer directly correlated to the amount of protein. Various fractions usually being expressed as a percentage of the total rather than in absolute amounts. The amount of dye bound by each fraction can be simply determined by cutting out the stained bands and eluting the dye into a fixed volume of a suitable solvent. The absorbance of each solution is measured and the sum of absorbance values assumed to be proportional to the total amount of protein. Hence the amount of protein in each fraction can be calculated as the percentage of the total.

14. 2. Alternative quantitative method Densitometer (modern electrophoresis) More satisfactory method of quantitation by scanning the stained electrophoretic strip using a densitometer, which is a modified photometer in which the electrophoretogram replaces the usual glass cuvette. The strip is slowly moved across the light path and the signal from the photoelectric detector is plotted by a pen recorder. The result is a trace that plots the absorbance value against the distance along the electrophoretogram and the area under the trace is proportional to the total protein content. From the area under each peak, the proportion of protein associated with that peak can be calculated.

15. Immunological methods Immunoblotting technique (Western blotting) Immunoblotting technique use antibodies (or other specific ligands) to identify target proteins among a number of unrelated protein species. The prerequisite is the availability of an antibody, either polyclonal or monoclonal, against the test protein.

16. The steps of immunoblotting After the initial separation by a conventional electrophoretic technique in a gel, the proteins are transferred (or blotted) electrophorecally from the gel to a membrane, usually nitrocellulose. The gel and the membrane, which has been previously soaked in a suitable electrophoretic buffer, are then sandwiched between two electrodes. A voltage is applied, e.g. 100 V, and the proteins migrate from the gel to the adjacent membrane. After about 1 h, the membrane is removed and carefully washed with buffer and a dilute solution of bovine albumin to block any subsequent, non-specific adsorption of antibodies to the membrane.

17. The steps of immunoblotting (cont) The next step involves treating the membrane with a suitable dilution of the specific antibody and allowing the reaction to take place for at least 1 h. Excess antibodies are then washed from the membrane and the bound antibody which remains is detected using a second antibody against the first, e.g. anti-rabbit immunoglobulin. The bands can then be visualized using an appropriate method. The resulting patterns of zones is then compared with the electrophoretogram prior to immunoblotting and the specific proteins pinpointed for any further investigation or separation.

18. Immunoaffinity purification Useful technique for the separation and purification of individual proteins due to the powerful immunogens and the availability of specific antibodies. Has been used to purify a wide range of proteins such as hormones, membrane receptor and complement proteins. The availability of suitable antibodies is essential and these may be raised by whole animal polyclonal techniques or by monoclonal cell culture. The former antibodies may need some prior purification before being immobilized.

19. Chromatographic methods Various chromatographic techniques may be applied to the study of protein mixtures. Gel permeation chromatography is frequently used to separate protein mixtures but it is necessary to have some prior knowledge regarding the size of the proteins present in order to select the most suitable gel. Ion-exchange chromatography using the substituted cellulose ion- exchangers is frequently used in the preparative aspects of protein analysis. Affinity chromatographic techniques, including those that employ antibodies as ligands, permit highly specific separations of proteins. Reverse-phase HPLC can be used for the separation of peptides and proteins. Hydrophobic interaction chromatography (HIC) is particularly useful for protein separations.

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