Disease Diagnosis

One of the most widely utilised analytical tools in Molecular Biology, Proteomics and disease diagnosis is the procedure and methods of electrophoresis. The principle of this procedure is that “electrophoresis is the migration of electrically charged particles or ions in solutions due to an applied electric field”1. The ability to separate substances which are almost identical including different proteins for analysis has increased throughout the past 7 decades.

This is contributed to the introduction of zone electrophoresis in paper and more recently in gels like polyacrylamide and agarose. Today the methods used in electrophoresis have increased our advances in biochemistry, molecular biology studies and diagnosis of disease, for example cancer. It has also become an invaluable tool in forensic science as it can identify species and individuals1. The field of Proteomics is “the large-scale screening of the proteins in a cell or an organism or biological fluids, a process which requires stringently controlled steps of sample preparation, 2-D electrophoresis, image detection and analysis, spot identification, and database searches” 2. The main core of the technology and advances in the study of proteomics is due to the application of 2D electrophoresis and as it stands at the moment there is no alternative method available for resolving thousands of proteins in one separation technique.

An Introduction to Two Dimensional Electrophoresis.

The most widely used tool in proteomics used to analyse proteins is the technique of Two-dimensional gel electrophoresis, which is often abbreviated as either 2DE or 2-D Electrophoresis. This is a two step process technique which separates proteins in accordance to two independent properties, there isoelectric point (pI) and there relative molecular weight (Mr) 2. In the first dimension the proteins or analytes will lie along a line separated by their pI, in 2DE the technique starts with 1DE then in the second dimension the are separated by gel electrophoresis in SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) which separates the proteins in a direction which is 90% from the first according to their Mr. Each spot that shows up on the resulting 2DE technique matches up to a single protein type in the sample.

This technique enables us to separate and gain information such as the pI and Mr of thousands of different proteins. The resulting picture shows that the proteins and analytes spread out across the 2-D surface instead of a straight line. This is because it is not very likely that any two proteins and analytes will have both properties of the same point or weight. However it is possible that they can be the same in one of the properties3. According to Vesterberg1 the first and earliest recorded use of 2-D gel electrophoresis was in 1956 in a paper that was published by Smithies and Poulik.

In there procedure they used starch gels, but these gels were not very optimal, however according to Righetti4 all the credit for the development and success of this technique went to O’Farrell and Klose in 1975, In this procedure the separation was obtained by pre-treating the samples in heated up SDS-urea solutions with the IEF (isoelectric focusing) followed by the SDS-PAGE. “he was able to resolve and detect about 1100 different proteins from lysed E. coli cells on a single 2D map and suggested that the maximum resolution capability might have been as high as 5000 different proteins” 4. The accuracy and effectiveness of 2DE as a protein separation technique has been extensively utilised since its introduction, but the significance of the ability of 2DE has only become apparent in the last few years due to the new developments in this procedure which have come about. When the introduction of immobilised pH gradients came about it brought superior resolution and reproducibility to the first step of the process IEF.

The technique which is now the most commonly used in which the carrier ampholyte generated pH gradients are now replaced by pH immobilised gradients and slabs supported by plastic backing has replaced the tube gels by Grg et al2. New techniques in mass spectrometry developed now enables us to get rapid identification of minute quantities of proteins taken from the spots on 2DE. We now have data available for a number of organisms genomes. This gives us the ability to also rapidly identify the gene that encodes a protein which has gone through 2DE separation. Today we often use computers and software based image analysis tools that analyse bio-markers and quantifying individual proteins. This software tool shows match spots between gels of similar samples and proteomic differences between early and advanced stages of illness.

Though this technique is commonly used it does have a few disadvantages. Some of the software used tends to agree on the quantification and analysis of well-defined well-separated protein spots; however they deliver different results and analysis tendencies with less-defined less-separated spots3. The largest and most used application of 2DE is in the analysis of the whole protein in the field of proteomics.

Due to its unparalleled ability to separate simultaneously thousands of proteins, this technique is used for the separation, identification and quantification of multiple proteins from a single sample. Other applications of 2DE in proteomics include as well as protein identification are cell differentiation, detection of disease markers, the monitoring of therapies, investigation and development in drug discovery, treatment and cure in cancer research. Principles of IEF and SDS PAGE.

The first step of 2DE is the step involving IEF and the investigation to discover and identify the pI of the samples tested. Depending on the pH of there surroundings proteins carry a positive, negative or a zero net charge and it leads to them being known as amphoteric molecules. The total amount of positive and negative charge of a proteins amino acid side chains and carboxyl terminal is known as the net charge of that protein. When the net charge of the protein is zero in a specific pH this is known as its pI. When the pH is higher than the pI the proteins will carry a negative charge and at a lower pH they will carry a positive charge.

When carrying out separation of a protein in a pH gradient and you introduce an electric field, the proteins will move along to the position in the gradient where its net charge is zero. Negatively charged proteins will migrate in the direction of the anode until it reaches the point of that individual proteins pI and the positively charged will move towards the cathode until they reach their pI. The proteins charge can be regained if it diffuses away from the pI and they will migrate back across the gradient. It is this that is the focusing effect of IEF which allows proteins to be separated on the basis of very small charge differences5. The second step technique is a common method for the separation of proteins using discontinuous polyacrylamide gels as a medium with SDS to denature the proteins. It is known as sodium dodecyl sulfate polyacrylamide gel electrophoresis or SDS PAGE.

As SDS is an anionic detergent the molecules hold a net negative charge throughout a wide range of pH. The SDS becomes bound to a polypeptide chain in accordance with its Mr, it is the SDS’s negative charge that destroys most of the proteins structure and is attracted to the positively charged electrode known as an anode in the electric field. The gel has varying sizes of pores throughout its structure that slow the larger molecules migrating and allows the smaller molecules to pass through faster during separation. SDS-PAGE is used to estimate relative molecular mass.

These two procedures are brought together to perform 2DE. The IEF is run at a current of 2mA for 30 minutes to allow the pH gradient to gain equilibrium. The current runs through for a further 30 minutes to achieve full separation of the proteins. The IPG strip or gel rods are then removed when the migration is complete. The gel rod is then transferred to the SDS PAGE after being treated in the buffer to achieve equilibration. This part of the method is run at 100-200 V until the front of the dye is 1cm from the edge of the slab. Using either a silver stain or coomassie blue the gel is processed for analysis5.

As well as advances in procedure from the method by Smithies and Poulik1 to the method pioneered by O’Farrell and Klose4 and the alternative to carrier ampholytes by G�rg et al2, methods in sample preparation have also seen advances and …

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Write an essay in up to 2000 words on the following topics, describing the procedures that can be used to isolate proteins from a cell and the methods involved in checking the purity. Using diagrams to help, you should explain …

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