SDS and 2D page
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SDS-PAGE is a common method to separate proteins based on their molecular mass. The proteins are first denatured by SDS, which coats each protein with a uniform negative charge. This causes the proteins to migrate towards the anode during electrophoresis, with smaller proteins moving faster through the discontinuous gel system. Two-dimensional gel electrophoresis combines isoelectric focusing, which separates proteins based on isoelectric point, with SDS-PAGE to allow better separation of proteins with similar masses.
SDS and 2D page
- 1. SUBMITTED BY Vivek kumar M.sc MICROBIOLOGY Bangalore university
- 2. SDS PAGE It is most common method for separation of proteins based on their molecular masses. This is a form of denaturing electrophoresis. The sample molecule are first denatured and then subjected to electrophoresis. The detergent SDS(Sodium dodecyl sulphate) is used. It consist of hydrophobic chain and polar sulphated head.
- 3. The hydrophobic chain can intercalate in to hydrophobic interior of the protein and disrupt its compact structure. SDS coats protein with a uniform negative charge and causes them to migrate to the anode. The net intrinsic charge of the protein is cancelled. The protein move with a velocity inversely proportional to their molecular mass. SDS page is done in discontinuous gel systems consisting of two gels: STACKING GEL and RESOLVING GEL
- 5. STACKING GEL It is of pH 6.9. Its purpose is to concentrate the sample components into this bands. The gel has a low percentage of acrylamide (3-5%) ,with low ionic strength.
- 6. RESOLVING GEL It has high percentage acrylamide (8-20%) with higher ionic strength and alkaline pH. This gel achieves separation of molecule as they move through it. Usually Tris-Glycine buffer is used.
- 8. ADVANTAGES Widely used due to its simplicity and versatility. Molecular mass of protein can be obtained.
- 9. DISADVANTAGES Two proteins having same molecular mass can not be separated. If protein deviate from ideal globular shape or if they are bound above or below average amount of SDS, inaccurate mass estimate will result. So it is necessary to use standard proteins to compare the molecular mass. Post translational modification also affect mobility of proteins.
- 11. ISO ELECTRIC FOCUSING Net charge of protein is highly influenced by the pH. At low pH, most proteins are positively charged. At high pH, they are negatively charged. The pH value at which proteins have no net charge is the isoelectric point(PI).
- 12. A stable pH is established in a solution using a mixture of ampholytes. Ampholytes are synthetic low molecular mass polymers having varying PI. When such a mixture is placed in an electric field they form a pH gradient. When a protein is mixed with pH gradient and subjected to electric field, it will migrate towards the PI.
- 13. At pH above PI it will have net positive charge and below PI it will have negative charge. The net charge is 0 and no electric mobility. This experiment is called IEF as it involves focusing of molecules at their individual PI values. IEF is usually conducted in glass tubes or in horizontal gels. Precast IEF gels are available. PI may be determined by running a mixture of known protein of known PI value.
- 15. 2-D PAGE Two proteins of same molecular mass can be separated. This is a combination of IEF and PAGE. Proteins are separated in first dimension by IEF. This is performed in 9M urea in rod shaped gels.
- 16. Then the IEF gel is placed along the tops of SDS gel and electrophoresis is done. Proteins separated in IEF is based on their PI value. 2nd dimension mainly on the basis of molecular mass. 2-D PAGE is based on the assumption that it is unlikely that 2 proteins will have same PI and molecular mass.