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Proteomics: Investigation Of Sample Loading Techniques For Iso-Electric Focusing

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Summary of the project

Complex protein mixtures extracted from cells, tissues or other biological samples can be separated and then identified. In this project, we are investigating different conditions that might influence how proteins are separated. It involves trying a combination of conditions to load complex protein samples onto special gel strips, which are used to separate proteins according to their iso-electric points (pI). An iso-electric point is the specific pH at which the net charge of a protein is zero. 1 After this, further separation is carried out, whereby proteins are separated according to their molecular weight. 1


To identify the various types of proteins that are present in a protein sample, the proteins must first be separated. The proteins are separated by first dimension isoelectric focusing (IEF), followed by second dimension sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

The protein sample is loaded onto an immobilised pH gradient (IPG) strip. IEF, an electrophoretic method, is then carried out. It separates proteins according to their isoelectric points (pI). Depending on the pH of their surroundings, proteins, being amphoteric molecules carry either positive, negative or zero net charge. 1 The sum of the negative and positive charges of its amino acid side chains and amino- and carboxyl- terminals makes up the net charge of a protein. 1The isoelectric point is the specific pH at which the net electrical charge of a protein is zero. 1

After IEF, second dimension separation is performed. SDS-PAGE separates polypeptides according to their molecular weights (MW). 1This technique is performed in polyacrylamide gels containing sodium dodecly sulphate (SDS). IEF and SDS-PAGE are both high-resolution techniques. 2 The resolution obtained during first dimension separation is not lost when the IPG strip is joined to the SDS-PAGE gel. 2


Two-dimensional gel maps facilitates the analysis of multiple proteins in parallel. It is, and will continue to be in the foreseeable future, very much an integral part of proteomics research. In order to allow for more in-depth analysis of proteins, there is a need to develop separations technology so as to increase the resolution of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). This can be achieved by investigating the factors affecting protein separation. Gel maps in the pH range of 6 - 11, which is the alkaline region, typically has poorer resolution due to incomplete separation of the protein thus resulting in streaking. 4

Our objectives are to combine conditions to load complex protein samples onto IPG strips for iso-electric focusing gel (IEF), coupled with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and to evaluate the results by image analysis after silver staining.

Our hypothesis is that using both cup-loading and including the sample in the rehydration buffer will produce a gel with better resolution, in terms of more localized spots.

2 experiments were conducted loading 200ug of sample NSO PFD3(3); experiment 1 using 200ug cup loading only and experiment 2 using 100ug rehydration loading followed by 100ug cup loading, to find out which sample loading method produced gels with better resolution. After that, first dimension IEF and SDS-PAGE and silver staining were carried out.

Experiment 1 was found to produce clearer and more distinct spots. Although experiment 2 produced a gel with less distinct spots, it can be further improved on by modifying the focusing time for IEF.

Background to first dimension iso-electric focusing (IEF) 1

In IEF, proteins are separated on the basis of isoelectric points (pI). This is the pH where the net charge of the protein is zero. Positively charged proteins have pH values below their pI and negatively charged proteins have pH values above their pI.

Having a pH gradient present is very important in IEF. Under the influence of an electric field, a protein will move to the position in the pH gradient where its net charge is zero. A protein with a positive net charge will move towards the cathode, becoming less positively charged as it moved through the pH gradient until it reaches its pI. Similarly, a protein with a negative net charge will move to the anode, becoming less negatively charged until it too reaches zero net charge. The focusing effect of IEF concentrates proteins at their pIs and allows proteins to be separated on the basis of very small charge differences.

The slope of the pH gradient and the electric field strength determines the degree of resolution. Hence, IEF is performed at high voltages. When proteins have reached their final positions in the pH gradients, there is minimal ionic movement in the system, resulting in a very low final current. Each type of protein molecule accumulates and focuses into a sharp band at its characteristic isoelectric point. IEF performed under denaturing conditions gives a higher resolution and the cleanest results.

Sample loading methods 1

The protein sample can be applied either by including it in the rehydration solution or by applying it directly to the rehydrated immobilized pH gradient (IPG) strip via sample cups. The differences between both methods can be explained in the table below.

Advantages of loading sample using cup-loading method Advantages of including sample in the rehydration solution

Proteolysis and other protein modification is prevented as sample is not left to rehydrate overnight, as done when the sample is included in the rehydration solution * Allows larger quantities of proteins to be loaded and separated

* Allows more dilute samples to be loaded

* Since there is no specific application point of the sample, it minimizes formation of precipitate at the application point that often occurs when loading with sample cups

* Technically simpler as it avoids problems such as leakage than can occur when loading with sample cups

Background to second dimension sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 1

SDS-PAGE further separates proteins on the basis of their molecular masses by electropheresis. Separation exclusively by molecular mass is possible when proteins are treated with both sodium dodecyl sulphate (SDS) and a reducing agent. SDS is an anionic detergent that denatures proteins by wrapping around the polypeptide backbone. SDS conceals



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