SDS-PAGE of Proteins

SDS-PAGE

Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (SDS-PAGE)

The method is based on - the separation of proteins according to size

Used – Separation of proteins 

          determine the relative molecular mass of proteins

SDS (CH₃-(CH₂)₁₀-CH₂OSO₃^- Na⁺) → is an anionic detergent

Buffer-

1.            β-mercaptoethanol – reduces/breaks disulphide bonds in protein tertiary structure & converted to linear primary structure
2. 
   
              SDS - denatures the protein
3.        bromophenol blue – is an ionisable tracking dye which use to monitor the electrophoretic run
4.       sucrose or glycerol - gives the sample solution density which helps to settle the sample at the bottom of loading well
5.       Tris- HCl – gives the solution its pH buffering capacity

loading dye - 

1. help to get know when to switch off
2. show how goodly separated.

negatively charged SDS molecules binds for every two amino acid residues in denatured liner polypeptide chain.

2 parts in the gel.

Stacking Gel	Separating/Resolving Gel
1st part of the gel	2nd part of the gel
pH is 6.8	pH is 8.8
Have larger pore size(4% acrylamide)	Have smaller pore size(10-15% acrylamide/polyacrylamide)
Comparatively short ( 0.8cm)	Comparatively long ( 5cm)

stacking gel

stacking gel - concentrate the protein sample into a sharp band before it enters the main separating gel.

This is achieved by using differences in ionic strength and pH between the electrophoresis buffer and the stacking gel buffer.

Negatively increases as follows in stacking gel-

glycinate ions < protein–SDS complexes < chloride ions (Cl^-)

so, in this protein-SDS complex is stacked between glycinate ions & chloride ions (Cl^-). Due to this called as stacking gel

(glycine isoelectric point is 5.95. so, glycinate ions have slight negativity.)

• Conductivity inversely proportional Field strength
• Conductivity  proportional to concentration

Separating/Resolving Gel 

When glycinate reaches the separating gel it becomes more fully ionized in the higher pH environment and its mobility increases

Due to this, negatively increases as follows in separating gel-

protein–SDS complexes< glycinate ions< chloride ions (Cl^-)

then,

•      the smaller the protein -  the more easily it can pass through the pores of the gel, 
•      large proteins - are successively retarded by frictional resistance due to the sieving effect of the gels.
•      Due to this protein are separate on the basis of their size.

•      When the dye reaches the bottom of the gel, the current is turned off
•      Stain with an appropriate stain solution (usually Coomassie Brilliant Blue) and then washed in destain solution
•     The destain solution -  removes unbound background dye from the gel, leaving stained proteins visible as blue bands on a clear background

Gels of 15% polyacrylamide are therefore useful for separating proteins in the range

M_r 100 000 to 10 000

·       A pure protein should give a single band on an SDS–polyacrylamide gel