Electrophoresis: SDS-PAGE

Main idea of sds electrophoresis are present in this slides that for which purpose and how to do

1. ELECTROPHORESIS Prepared by Fakhre-yar Electrophoresis is a technique used in laboratories in order to separate macromolecules based on size. The technique applies a negative charge so proteins move towards a positive charge. The separation of macromolecules in an electric field is called  electrophoresis.

2. SDS-PAGE Sodium Dodecylesulfate Polyacrylamide Gel Electrophoresis Introduction: Standard test that used to determine the charged molecules, mainly the proteins and nucleic acids. Widely used in biochemistry and forensics, genetics and molecular biology. Laemmli system of SDS-PAGE was first introduced in 1970s.

3. SDS-PAGE Principle: Separate protein in an electric field. Migrates through a liquid or semisolid medium when subjected to an electric field from anode to cathode terminal. Molecules flow at different rates depends on the molecular size of proteins.

4. What is SDS -ively charged detergent. Used to denature and linearize the proteins. Coated the proteins with –ively charged.

5. What is PAGE SDS-PAGE is differentiated into two systems Continuous sds-page Discontinuous sds-page Polyacrylamide is used to form a gel, a matrix of a pores which allow the molecules migrate at different rates.

6. Polyacrylamide gel The size of pores is determine by the concentration of acrylamide. The higher the concentration, the smaller the size of pores. Discontinuous sds-page consists of two different gel. Stacking gel (top gel) -4% of acrylamide Separating gel (bottom gel) range from 5-15% of acrylamide.

7. Why polyacrylamide used for a gel Chemically inert Electrically neutral Hydrophilic Transparent for optical detection

8. Preparation of gel Clean the plates and combs. Set-up the plates on the rack. Pour the separating gel. Pour the stacking gel. Gel storage.

9. Process of SDS-PAGE Boil the sample for 10 mints' to completely denature the proteins. Assemble the gel into the apparatus. Pour the buffer solution into the chamber. Load 20µl of sample into the well. After that, run electrophoresis by connecting the current supplies.

10. Visualization of protein bands Visualizes the band under UV light Types of stain: Coomssie Blue Traditional method requires staining followed by distaining to remove background gel staining. Most common and least sensitive. Limited to ˜ 100ng of protein. Silver stain Most sensitive test. Detection limit 0.1-1.0ng of protein.

11. Applications Determine purity of protein samples. Determine molecular weight of proteins. Identifying disulfide bonds b/w proteins. Quantifying protein. Blotting applications.