Lecture 04.ppt

Unit III : Chromatography and Electrophoresis Biological and Biochemical Techniques BBT-304 Dr. Subhash Kudale Electrophoresis Lecture IV : SDS-PAGE Electrophoresis SDS-PAGE Electrophoresis - SDS PAGE Electrophoresis – SDS-P...

Unit III : Chromatography and Electrophoresis Biological and Biochemical Techniques BBT-304 Dr. Subhash Kudale Electrophoresis Lecture IV : SDS-PAGE Electrophoresis SDS-PAGE Electrophoresis - SDS PAGE Electrophoresis – SDS-Page: Sodium Dodecyl Sulfate (SDS) polyacrylamide gel electrophoresis is mostly used to separate proteins accordingly by size. This is one of the most powerful techniques to separate proteins on the basis of their molecular weight. Principle: This technique uses anionic detergent Sodium Dodecyl Sulfate (SDS) which dissociates proteins into their individual polypeptide subunits and gives a uniform negative charge along each denatured polypeptide. It forces polypeptides to extend their conformations to achieve simi­lar charge: mass ratio. SDS treatment there­fore eliminates the effects of differences in shape so that chain length, which reflects their molecular mass, is the sole determinant of migration rate of proteins in the process of elec­trophoresis. 3 Lecture IV – SDS-PAGE electrophoresis SDS-PAGE Electrophoresis - When these denatured polypep­tides are loaded at the cathode end of an elec­trophoretic tank having polyacrylamide gel (as the supporting media) and subjected to an elec­tric field, then we get clear bands of proteins arranged in an decreasing order of their mo­lecular mass from the cathode to anode. The rate of movement is influenced by the gel’s pore size and the strength of electric field. In SDS- PAGE the vertical gel apparatus is mostly used. Although it is used to separate proteins on a routine basis, SDS-PAGE can also be used to separate DNA and RNA molecules. 4 Lecture IV – SDS-PAGE electrophoresis SDS-PAGE Electrophoresis - 5 Lecture IV – SDS-PAGE electrophoresis SDS-PAGE Electrophoresis - Instrumentation: Electrophoretic Apparatus: Vertical horizontal tank with electrodes, gel cas­settes, Teflon spacers, clips, pipette or sy­ringe, comb, acrylic cover. Power Supply: A power supply of 100-200 volts is needed. This is ideal for run­ning and transferring protein resolving gels. Staining Box: These are trays in which the gels are stained and made up of clear plastics. These are resistant to most or­ganic dyes, silver and other stains. 6 Lecture IV – SDS-PAGE electrophoresis SDS-PAGE Electrophoresis - Supporting Media: SDS-PAGE acrylamide is used as the supporting medium. It is a white crystalline powder, when acrylamide dissolves in water, it undergoes polymerization reaction to form a net-like structure called polyacrylamide. Polyacry­lamide is a polymer (CF2CHCONH2-) formed from acrylamide subunits that can also be readily cross-linked. This type of electro­phoresis has a discontinuous system of gel, i.e., we have two different systems of gels present in the electrophoretic tank physi­cally placed one over another. Resolving Gel: This is also called sepa­rating or running gel. The separating gel constitutes about 2/3rd of the length of gel plate and is prepared by 5-10% of acrylamide. The pores in this gel (which is formed after the polyacrylamide is cross- linked) are numerous and smaller in dia­meter which impacts sieving property to this gel. 7 Lecture IV – SDS-PAGE electrophoresis SDS-PAGE Electrophoresis - Stacking gel: Stacking gel is poured on the top of resolving gel and a gel comb is inserted which forms the well. It is the up­per layer of gel and constitutes 1/3rd of the gel plate. The percentage of acrylamide is chosen depending on the size of protein that one wishes to identify or probe in the sample. The smaller the known weight, the higher the percentage that should be used. Generally, the percentage of acrylamide in stacking gel is 2-5%. It is highly porous and devoid of molecular sieving action. Buffer: Two types of buffers are used in SDS-PAGE. The lower reservoir (which has the running gel) has amine buffers. It is adjusted by using HCl. The upper re­servoir (which has stacking gel) also has amine buffers but its pH is slightly above that of running gel buffer and is adjusted with glycine instead of HCl. 8 L Lecture IV – SDS-PAGE electrophoresis SDS-PAGE Electrophoresis - Stains: The stains are used to see the bands of separated proteins after the pro­cess of electrophoresis. Comassie Bril­liant Blue R-250 (CBB) is the most popu­- lar protein stain. It is an anionic dye, which binds with proteins non- specifically. Pro­teins in the gel are fixed by acetic acid and simultaneously stained. The excess dye in­corporated in the gel can be removed by de-staining with the same solution but without the dye. The proteins are detected as blue bands on a clear background. 9 Lecture IV – SDS-PAGE electrophoresis SDS-PAGE Electrophoresis - Procedure: The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent, an an­ionic detergent which denatures secondary structure. Besides addition of SDS, proteins may optionally be boiled in the presence of a reducing agent, such as Di-Thio-Threitol (DTT) or 2-mercaptoethanol, which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary pro­tein folding, and breaking up quaternary pro­tein structure (Oligomeric subunits). This is known as reducing SDS-PAGE, and is most commonly used. Non- reducing SDS-PAGE (no boiling and no reducing agent) may be used when native structure is important in further analysis (e.g., enzyme activity, shown by the use of zymograms). The denatured proteins are subsequently loaded into the wells of stacking gel flooded with stacking buffer. 10 Lecture IV – SDS-PAGE electrophoresis SDS-PAGE Electrophoresis - This end is connected with the cathode of power supply. Then an electric current is applied across the gel, causing negatively charged proteins to migrate across the gel towards anode. After crossing the stacking gel, denatured proteins enter the running gel which has its own buffer system (running buffer). Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty. After the sepa­ration is over the gel is gently taken out and transferred to the staining box and treated with the staining dye, e.g., CBB R-250. Excess of stains are removed by de-staining using ace­tic acid solution. The bands appear to be blue stained which are then analysed according to the need of the experiment. 11 Lecture IV – SDS-PAGE electrophoresis SDS-PAGE Electrophoresis - Application: Establishing protein size Protein identification Determining sample purity Quantifying proteins Blotting applications 12 Lecture IV – SDS-PAGE electrophoresis SDS-PAGE Electrophoresis - Advantages of SDS-PAGE: Mobility of the molecules is high and sepa­ration is rapid. All the proteins are negatively charged; therefore, all migrate towards anode. The proteins treated with SDS fixed dyes are better than the native proteins. SDS solubilizes all proteins, including very hydrophobic and even denatured proteins. 13 Lecture IV – SDS-PAGE electrophoresis Thank You