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

1. • 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. 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.

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

6. 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.

7. 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.

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

9. 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.

10. 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.

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

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