640_chp7 - 1 protein variation • how do we measure...

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Unformatted text preview: 1 protein variation • how do we measure variability? – structural similarity? how? • immunology • enzyme activity • indirect structural differences (electrophoresis) • amino acid similarity (sequence) DETECTION OF PROTEIN POLYMORPHISM BY ELECTROPHORESIS • Separation of components by: – size – shape – net charge PROTEIN ELECTROPHORESIS • developed as biochemical techniques in late 1930’s for separation of peptides moving boundary electrophoresis PROTEIN ELECTROPHORESIS • continued development in 1940’s for biochemical separation of peptides • application to sickle cell hemoglobin to show first “molecular disease” (1948) PROTEIN ELECTROPHORESIS • 1950’s: development of solid support media – more reliable separation and characterization • starch • cellulose acetate • polyacrylamide gel electrophoresis ( PAGE ) 2 PROTEIN ELECTROPHORESIS • electrophoretic techniques developed without any thought concerning population studies – biochemical methods for protein separation – how about using this technique to study populations? • what are the characteristics of an ideal set of loci to use to study populations? – co-dominant inheritance – random choice of loci to estimate average variation • loci not picked because they are known to be variable – many loci can be studied from same individuals how do we study protein polymorphism • consider a tissue sample – contains many different protein – structural proteins • actin, tubulin – soluble cytosolic proteins • enzymes – membrane bound • enzymes, receptors how do we study protein polymorphism how do we study protein polymorphism • Or, consider a sample of blood/serum – serum contains many different soluble proteins • albumin, transferrin, ceruloplasmin, etc.,etc., etc . how do we study protein polymorphism • Or, consider a sample of blood/serum – serum contains many different soluble proteins • albumin, transferrin, ceruloplasmin, etc.,etc., etc . – red blood cells • proteins/enzymes similar to many other cells • hemoglobin (>50% of RBC protein content) how do we study protein polymorphism • Or, consider a sample of blood/serum – serum contains many different soluble proteins • albumin, transferrin, ceruloplasmin, etc.,etc., etc . – red blood cells • proteins/enzymes similar to many other cells • hemoglobin (>50% of RBC protein content) – white blood cells • additional cell specific proteins 3 how do we study protein polymorphism • most electrophoresis uses denaturing conditions – SDS or other denaturing agents or heat – use general protein staining • not applicable to most proteins; depends on concentration how do we study protein polymorphism • try non-denaturing conditions – preserve activity of proteins – allows the detection of proteins independent of concentration in sample – dependent only on available histochemical staining procedures • exploit available histochemical staining...
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640_chp7 - 1 protein variation • how do we measure...

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