LIST OF REFERENCES

1) SDS-PAGE; Facts, Discussion Forum, and encyclopedia article. Retrieved on October 12, 2009 from http://www.absoluteastronomy.com/topics/SDS-PAGE

2) SDS-PAGE gel electrophoresis. (2008). Molecular station. Retrieved on October 12, 2009 from http://www.molecularstation.com/sds-page-gel-electrophoresis/

3) Saviana, B., Pons, L., Namour, F., Quilliot, D., Ziegler, O. and Guéant, J.L. (2000). Sodium dodecyl sulphate gel electrophoretic preparation of protein standard human apolipoprotein B-48. Journal of chromatography B: Biomedical sciences and applications, Vol, 742, issue 2, 421-426.

4) Dr. William H. Heidcamp. Chapter 4: Electrophoresis-Introduction. Biology Department, Gustavus Adolphus College. Cell biology laboratory manual.

5) http://www.geneed.com

QUESTIONS

1) Separation of proteins by gel electrophoresis is based on:
a) Relative size of proteins
b) Mobilities or migration rates in a charged electrical field
c) Charged species
d) Hydrophobicity of proteins

2) Which of the following statement about gel electrophoresis is FALSE?
a) Sodium dodecyl sulphate is added to the protein sample so as to give the proteins a net negative charge
b) Once the power supply is switched on, the proteins loaded into the wells of the polyacrylamide gel will migrate from cathode to anode
c) Samples are loaded into the stacking gel and separation of proteins only takes place in the resolving gel
d) Tracking dyes are often used to monitor migration of proteins

3) The percentage of acrylamide in the resolving gel is usually lower than the stacking gel so as to allow the proteins to migrate slower hence allowing better separation of the proteins
a) True
b) False

4) Molecular weight of an unknown protein maybe determined from gel electrophoresis by
a) Measuring the distance travelled from the well to the protein band
b) Measuring the Rf value and comparing to that of the molecular weight (MW) markers
c) Measuring the Rf value and obtaining the MW from the calibration plot of MW versus Rf values of standard markers
d) Measuring the Rf value and obtaining the MW from the calibration plot of In MW versus Rf values of standard markers

5) The non-denaturing gel electrophoresis may separate proteins based on its charge and size
a) True
b) False

6) Techniques that can be used to detect proteins separated by SDS-PAGE are as follows:
a) Radiolabelled proteins maybe pressed against a sheet of X-ray film
b) Coomassie blue-250
c) Silver staining
d) Heavy metal staining
e) All the above

7) In the preparation of polyacrylamide gel, the catalyst that is used to accelerate polymerization and cross linking of gel is
a) TEMED
b) APS (ammonium persulphate)
c) Acrylamide
d) Bisacrylamide
e) No answer

8) What are the application of SDS-PAGE gel electrophoresis
a) To get a DNA fingerprint for forensic analysis
b) To get a DNA fingerprint for paternity testing
c) To get a DNA fingerprint to understand the evolutionary relationships among organisms
d) To test for genes associated with a particular disease.
e) All the above


The answers: a, d, a, b, a, e, a, e

ANIMATION

APPLICATIONS

Gel electrophoresis is used in DNA fingerprinting which has applications in forensics. Certain DNA segments are characteristic and vary from person to person. These segments are cut at recognition sites by restriction endonuclease enzymes and run on gel by electrophoresis. The position and number of bands on each lane of gel is characteristic of the DNA sample and considered as the genetic "fingerprint" of that sample.

Example of gel electrophoresis usage;

1. To get a DNA fingerprint for forensic analysis
2. To get a DNA fingerprint for paternity testing
3. To get a DNA fingerprint to understand the evolutionary relationships among organisms
4. To check a PCR reaction.
To test for genes associated with a particular disease

ELECTROPHORESIS and STAINING

The denatured proteins are placed at one end of the polyacrylamide gel layer; submerged in a suitable buffer. Then, an electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the anode. Hereby, depending on their size, each protein will move in various distances through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty as they encounter more resistance. After, a few hours, the proteins can be measured, where it differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin.

Following electrophoresis, the gel can be stained (most commonly with Coomassie Brilliant Blue or silver stain), in order to visualize the separated proteins. After staining, different proteins will appear as distinct bands within the gel therefore, it is common to run molecular markers of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker.

Gel electrophoresis is usually the first choice as an assay of protein purity due to its reliability and ease. The presence of SDS and the denaturing step causes proteins to be separated solely based on size. False negatives and positives are possible. A co-migrating contaminant can appear as the same band as the desired protein. This co-migration could also cause a protein to run at a different position or to not be able to penetrate the gel. This is why it is important to stain the entire gel including the stacking section. Coomassie Brilliant Blue will also bind with less affinity to glycoproteins and fibrous proteins, which interferes with quantification.

PROCEDURE

Resistance provided by the matrix is in relation to the molecular size and shape of the molecules moving through it. The greater molecule move slowly and smaller molecule migrate fast. However, there has a problem where the race pitting a large and highly charged molecule against a small, more neutral molecule that will be unfaired. By make sure that the ratio of charge and mass is constant, we need to eliminate the differences between in charge/mass ratio and differences in shape. Thus, by mixing the protein with the ionic detergent sodium deodecyl sulfate (SDS), the proteins become coated with negative charges, unfolding into a rod, a conformation that maximally separates those mutually repellent negative charges.

MATERIALS

1. Power supply


2. Gel electrophoresis set


3. Buffer

Gel electrophoresis set:

• Comb


• Gel tray


• Gel chamber

Buffer:

• Tris-Borate EDTA (TBE) at 1x or 0.5x


• Tris-Acetate EDTA (TAE) at 1x


• Function of buffer: maintain pH by increasing ion H+ concentration

STACKING GEL and RESOLVING GEL

Staking gel or spacer gel: a large pore poly acrylamide gel (4%) which is prepared with Tris buffer pH 6.8 of about 2 pH units lower than that of electrophoresis buffer. These conditions basically provide an environment for Kohlrausch reactions, as a result, proteins are concentrated to several folds and a thin starting zone of the order of 19 microns is achieved in a few minutes. This gel is cast over the resolving gel. The height of the staking gel region was always maintained more than double the height and the volume of the sample to be applied.

Resolving gel or Separating Gel: A small pore polyacryl amide gel (3 - 30%) which is prepared using Tris buffer used is of pH 8.8. In this gel, macro molecules separate according to their size. Normally in experiment, 8% gel for separate 24 – 205 kDa proteins, 10% gel for separate 14-205 kDa proteins and 12% gel for separate 14-66 kDa proteins.

ROLE OF SDS

Sodium lauryl sulfate, in science referred to as sodium dodecyl sulfate (SDS), is commonly used in preparing proteins for electrophoresis in the SDS-PAGE technique. Generally, the solution of proteins that is required to be analyzed is first mixed with sodium-dodecyl sulfate. When the proteins are treated with SDS, the polypeptide become rods of negative charge with equal charge densities or charge per unit length, and their separation is a function of the size differences only. When one never treat the proteins with SDS, different proteins with similar molecular weights would migrate differently due to differences in mass charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure. This is basically known as Native PAGE. Thereby, adding SDS could solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide.

In additional, SDS bind in a ratio of approximately 1.4 g SDS per 1.0 g protein (although binding ratios can vary from 1.1-2.2 g SDS/g protein), giving an approximately uniform mass:charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to the size of the protein only. Besides that, a tracking dye may be added to the protein solution so that the progress of the protein solution through the gel during the electrophoretic run can be monitored.

THEORY OF SDS-PAGE

Firstly, the proteins molecules are exposed to ionic detergent SDS (sodium dodecylsulfate) before or during the gel electrophoresis, where this SDS will then denatures the proteins and binds to the uncoiled molecules.

Generally, SDS binding confers a negative charge on all the proteins, hence in an electric field proteins will migrate solely as a function of their molecular weight. Hereby, can be illustrate that, the SDS treatment eliminates the effects of differences in shape so the mass plays the vital role in protein separation. SDS is very significant as it can separate chain that differ in molecular weight by less than 10 percent, and the molecular weight of that particular protein can be measured by comparing with the protein ladder or molecular weight ladder which is run on the same gel at the other lane.

In addition, it is a technique separating macromolecules such as DNA, RNA, and proteins. It is widely used for displaying proteins, for generating Western blots, and for separating nucleic acids that differ in length. The condition for separating by size the whole spectrum of typical cellular polypeptides, recommended from about 10, 000 to more than 100, 000 Da.

In general, electrophoresis is the process of applying an electric field to move charged molecules through a solution. The essence of this technique;

The mobility of a charged molecule = Net charge / Resistance of the solution through which it is moving

INTRO

SDS-PAGE is an abbreviation for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). It is one of the tremendous molecular biology techniques, which is used to separate proteins molecules accordingly to its size.

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SSG 3162
TECHNIQUE AND MOLECULAR
BIOLOGY ASSIGNMENT

PREPARED BY:

Renuka a/p P. Krishnan (AS070491) &

Noor Syahadah Tukimin (AS070321)

LECTURER:
Dr. Salehhuddin Hamdan


Company:
3rd Year Students of Faculty of Bioscience and Bioengineering,
Universiti Teknologi Malaysia