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

Protein gel electrophoresis

Polyacrylamide gel electrophoresis

TEMED - catalyzes free radical formationAPS - free radical donorBisacrylamide - crosslinking agent (19:1 ratio of acrylamide to bis maximizes crosslinking)

Higher % of gel - smaller pores (holes) so smaller fragments can be resolved

Protein gel electrophoresis

Gradient gels - As proteins migrate through increasing acrylamide concentration, smaller pores, mobility decreasesOnce proteins reach their “pore limit” little movement and can calc MW

Protein gel electrophoresis

Agarose gel electrophoresis

Agarose is a long sugar molecule

Room tempHeated, >70˚ C

Agarose - separation of large molecules8 kD to 800,000 kD

Polyacrylamide - separation of smaller molecules0.2 kD to 500 kD

Protein gel electrophoresis

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 gelStain gel with Coomassie Blue (binds to proteins)

SDS forms micelles and binds to proteins

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.

Protein gel electrophoresis

Separation of hemoglobin proteins

Hemoglobin - involved in oxygen transport in body

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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

Genetic disease in which person inherits gene for sickle-cell Hb from both parents

Hb V H L T P E E KHb-sickle V H L T P V E K

Hb-sickle is deoxygenated, insoluble and forms polymers that aggregateValine has hydrophobic side chain, glutamate has negative chargeValine creates sticky hydrophobic contact point where deoxy-Hb-sickle molecules associate forming long, fibrous aggregates

HemoglobinSickle Cell Anemia

Symptoms: weak, dizzy, short of breath, heart murmurssickle cells fragile - anemiacapillaries blocked -abnormal organ function

Patients with sickle cell anemia have to have inherited 2 copies of mutant geneInherit only 1 copy - resistance to malaria

Sickle cell trait ~10% of African American population

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

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)