1. Molecular Weight Determination
SDS-PAGE

2. Electrophoresis Movement of particles by an electric force.
Gel is the matrix perfused by buffer salts
Matrix will
reduce effects of diffusion
stabilize the system against turbulence (thermal agitation)
Introduce sieving action that allows separation based on molecular size
Buffer salts will
prevent sample interactions
create more conductivity
generates heat
Mobility = applied voltage x net charge on the molecule / function of molecule (shape, size and other physical matter)
Force: Total electric field is the effective charge and field strength
Opposing force is friction and pore size
When charge/mass is constant then large molecules move slower than small molecules.

3. Electrophoresis Two other factors are important
Ionic strength of surrounding solution
Buffer ions –
high ionic strength reduces effective charge of sample and temperature will increase;
low ionic strength slows the movement but has sharper peaks.
Best is Tris borate buffer – only few ions are free and large size ions reduce excessive heat generation
Temperature of the gel
Effects the viscosity of the gel
Need to run at constant power
SDS PAGE maintain high temperature to prevent renaturation

4. Electrophoresis Electric parameters:
Power = I2R I = current R= resistance
Buffer ions: cations move towards negative electrode in upper chamber and anions move towards positive electrode in the lower chamber.
Apparatus and buffers provide constant resistance.

5. Type of electrophoresis
Solution: Solutes dissolved in a solvent and electric force is applied.
Zonal: Aqueous ionic solution is carried in solid matrix.
Paper
cellulose acetate and cellulose nitrate
Gels
Starch gels – organelles
Agarose - DNA
Polyacrylamide – proteins and nucleic acids

7. Polymerization
Polyacrylamide gels are formed by the polymerization of acrylamide in aqueous solution in the presence of small amounts of a bifunctional crosslinker, usually bisacrylamide (bis).
The copolymerization of acrylamide with bisacrylamide produces a mesh-like network in three dimensions, consisting of acrylamide chains with interconnections formed from the bisacrylamide.

8. Polyacrylamide gels
T -the total percentage concentration (w/v) of monomer (acrylamide plus crosslinker) in the gel.
C - the percentage of the total monomer represented by the crosslinker.
For example, an 8%, 19:1 (acrylamide/bisacrylamide) gel would have a T value of 8% and a C value of 5%.

9. Polymerization
The polymerization of acrylamide and bisacrylamide occurs via a free-radical mechanism after adding a catalyst.
ammonium persulfate initiates free radicals and
tertiary aliphatic amine N,N,N',N'-tetramethylethylenediamine (TEMED) is a catalyst for free radical propagation

10. Polymerization
The presence of excess oxygen will inhibit the polymerization elongation process and can lead to shorter average chain length.
Deaeration under vacuum with a magnetic stirrer is needed prior to addition of initiators.

11. Advantages of Polyacrylamide Gels
Small pore size.
By changing the concentration of the monomer and the cross-linker, the pore size can be varied in a reproducible manner.
It is synthetic so no batch to batch differences.

12. SDS-PAGE
SDS (Sodium dodecyl sulfate) – PAGE (Poly Acrylamide Gel Electrophoresis) separation of proteins is based on molecular weight.
In their native form, proteins fold into a variety of shapes, some compact, some elongated. The rate of migration of native proteins through a sieving medium is therefore more a reflection of their relative compactness, and less an accurate measure of molecular weight.
Denaturing the proteins nullifies structural effects on mobility, allowing separation on a true charge/mass ratio basis. It also separates subunits in multimeric proteins, allowing analysis of large, complex aggregates.

13. SDS SDS is amphiphatic surfactant, an anionic detergent (pH above 7)
Hydrophobic tail binds to protein backbone
Polar heads denature ionic bonds
Uniform coating leading to charge/mass ratio consistent between proteins. 1.4g SDS/gram of protein is needed.

14. SDS-PAGE 2-mercaptoethanol – dissociates any disulfide bonds
SDS and urea disrupts ionic, hydrogen and hydrophobic bonds.
Overall – proteins have negative charge and disrupted secondary and tertiary charges.

16. SDS-PAGE
Protein now have negative charge. When electric field is applied they move towards cathode.
Larger the size of the protein, the more it is retarded by contact with the meshwork of acrylamide in the gel, so the smallest peptides will move farther in the gel and the larger molecules move progressively shorter distances

17. Discontinuous gel Electrophoresis
Two layers of different concentrations of gel and pH
Stacking low concentration and low pH
Separating gel – higher concentration of gel and high pH
Concentrate proteins in narrow band in stacking gel before traveling through separation gel for greater resolution of bands
Made up of stacking gel pH 6.8; separation gel, pH 8.8 and the tank .

18. Discontinuous Gels
Stacking gel has low concentration of gel, so ions move freely under applied voltage.
Glycine and chloride ions are used
Chloride ions move faster (leading ions) than glycine (trailing ions) and create steep voltage gradient. Glycine ions are pulled due to the charge gradient and the two ions move at same speed. The proteins with intermediate mobility are carried along becoming stacked into very thin, distinctive layers in order of mobility.
Resolving gel has high concentration of gel and high pH:
When the interface between the stacking and separation gel is reached by moving, the pH changes abruptly.
Glycine moves quickly and unstacks the proteins.
The movement of proteins is influenced by sieving capacity of the gel and its molecular weight because pH and the voltage are constant.

19. SDS-PAGE
After electrophoresis, the gel is treated with trichloroacetic acid solution
makes the proteins insoluble and
prevent them from diffusing during staining procedure

20. SDS-PAGE Staining to make invisible proteins visible.
Comassie blue in methanol-acetic acid

21. Procedure Prepare Tank buffer 2x treatment buffer Staining solutions
Destaining solutions I and II

22. Protein Samples
Dissolve protein sample with 2x treatment buffer (protein conc. 50 µg). Boil for 5min to denature.
Standards:
Lactalbumin; trypsin inhibitor; trypsinogen; carbonic anhydrase; albumin egg; albumin bovine

23. SDS-PAGE Preparing standard curve
Rf = distance traveled by peptide / distance traveled by tracking gel
Plot a standard curve of log molecular weight Vs Rf