1. Protein gel electrophoresis
• used to separate proteins based on a number of characteristics
Denaturing gel electrophoresis
separate by size
Nondenaturing (native) gel electrophoresis
separate by size, shape, charge

2. Protein gel electrophoresis
Polyacrylamide gel electrophoresis
TEMED - catalyzes free radical formation
APS - free radical donor
Bisacrylamide - crosslinking agent (19:1 ratio of acrylamide to bis maximizes crosslinking)
Higher % of gel - smaller pores (holes) so smaller fragments can be resolved

3. Protein gel electrophoresis
Gradient gels - As proteins migrate through increasing acrylamide concentration, smaller pores, mobility decreases
Once proteins reach their “pore limit” little movement and can calc MW

4. Protein gel electrophoresis
Agarose gel electrophoresis
Agarose is a long sugar molecule
Heated, >70˚ C
Room temp
Agarose - separation of large molecules
8 kD to 800,000 kD
Polyacrylamide - separation of smaller molecules
0.2 kD to 500 kD

5. Protein gel electrophoresis

6. Protein gel electrophoresis
SDS Gel Electrophoresis (agarose or polyacrulamide)
Denaturing condition
Denature protein by adding SDS (then separate by size only)
Used to estimate purity and molecular weight, separate proteins by size Electrophoresis of SDS-solvated protein on polyacrylamide gel
Stain gel with Coomassie Blue (binds to proteins)
SDS forms micelles and binds to proteins

7. Protein gel electrophoresis
Native gel electrophoresis
• polypeptides retain their higher-order structure and often retain enzymatic activity and interaction with other polypeptides
• migration of proteins depends on many factors, including size, shape, and native charge.
• native gels omit the SDS and reducing agent (DTT)
• do not put SDS or DTT in the sample buffer
• do not heat the samples
• prepare the gel and tank buffer solutions without SDS.

8. Protein gel electrophoresis
Separation of hemoglobin proteins
Hemoglobin - involved in oxygen transport in body
Normal adult Hb (Hb A) - two  subunits and 2  subunits
Sickle trait hemoglobin (Hb AS) - only one inherited mutation, 50% Hb S and 50% Hb A
Sickle hemoglobin (Hb S) - Glu -> Val mutation, Hb S inherited from both parents
When Hb S is deoxygenated it crystallizes in RBCs which leads to distortion of red cells and reduction in number of RBCs

9. Hemoglobin Sickle Cell Anemia
Genetic disease in which person inherits gene for sickle-cell Hb from both parents
Hb V H L T P E E K
Hb-sickle V H L T P V E K
Hb-sickle is deoxygenated, insoluble and forms polymers that aggregate
Valine has hydrophobic side chain, glutamate has negative charge
Valine creates sticky hydrophobic contact point where deoxy-Hb-sickle molecules associate forming long, fibrous aggregates
Symptoms: weak, dizzy, short of breath, heart murmurs
sickle cells fragile - anemia
capillaries blocked -abnormal organ function
Patients with sickle cell anemia have to have inherited 2 copies of mutant gene
Inherit only 1 copy - resistance to malaria
Sickle cell trait ~10% of African American population

10. Hemoglobin Abnormal Hbs detected in lab by electrophoresis
Hb at pH 9.2 has a net (-)charge so moves in electric field toward (+) electrode
pI of normal Hb is 6.9 but changes with mutations
In Hb S Glu -> Val ( chain), so 2 fewer (-) charges
Example of variations in migration on a gel when Hb mutations present

11. Sickle Cell Anemia
Examine electrophoretic behavior of Hb A, Hb S, Hb AS
1. Set up protein gel (gels are premade)
2. Load Hb samples
3. Separate Hb proteins by applying electric field
4. Take gel apparatus apart
5. Stain gel in Coomassie Blue (entire gel is blue at first)
6. Destain gel (removes nonspecific staining to reveal protein bands on gel)