Presentation on theme: "Protein gel electrophoresis"— Presentation transcript:
1 Protein gel electrophoresis • used to separate proteins based on a number of characteristicsDenaturing gel electrophoresisseparate by sizeNondenaturing (native) gel electrophoresisseparate by size, shape, charge
2 Protein gel electrophoresis Polyacrylamide gel electrophoresisTEMED - 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
3 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
4 Protein gel electrophoresis Agarose gel electrophoresisAgarose is a long sugar moleculeHeated, >70˚ CRoom tempAgarose - separation of large molecules8 kD to 800,000 kDPolyacrylamide - separation of smaller molecules0.2 kD to 500 kD
6 Protein gel electrophoresis SDS Gel Electrophoresis (agarose or polyacrulamide)Denaturing conditionDenature 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
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 proteinsHemoglobin - involved in oxygen transport in bodyNormal adult Hb (Hb A) - two subunits and 2 subunitsSickle trait hemoglobin (Hb AS) - only one inherited mutation, 50% Hb S and 50% Hb ASickle hemoglobin (Hb S) - Glu -> Val mutation, Hb S inherited from both parentsWhen 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 parentsHb V H L T P E E KHb-sickle V H L T P V E KHb-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 aggregatesSymptoms: weak, dizzy, short of breath, heart murmurssickle cells fragile - anemiacapillaries blocked -abnormal organ functionPatients with sickle cell anemia have to have inherited 2 copies of mutant geneInherit only 1 copy - resistance to malariaSickle 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 (+) electrodepI of normal Hb is 6.9 but changes with mutationsIn Hb S Glu -> Val ( chain), so 2 fewer (-) chargesExample of variations in migration on a gel when Hb mutations present
11 Sickle Cell AnemiaExamine electrophoretic behavior of Hb A, Hb S, Hb AS1. Set up protein gel (gels are premade)2. Load Hb samples3. Separate Hb proteins by applying electric field4. Take gel apparatus apart5. Stain gel in Coomassie Blue (entire gel is blue at first)6. Destain gel (removes nonspecific staining to reveal protein bands on gel)
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