Protein Primary Sequence Protein analysis road map: Bioassay design Isolation/purification Analysis Sequencing

Bioassay: Tracking a Target Protein A unique protein property for rapid, reproducible, sensitive and inexpensive identification Examples include: spectrophotometric detection of substrate/product, radioisotopic labeling for product detection, enzyme-linked immunosorbent assay (ELISA)

Differential Centrifuga- tion for Organelle Separation Marker enzymes confirm fractional cuts

(NH 4 )SO 4 Fractionation Desalting via dialysis or spin column

Gel Filtration Chromatography Separation based on size (size exclusion chromatography)

Ion Exchange Chromatography Cation resin: Carboxymethyl (CM) Anion resin: Diethyl- aminoethyl (DEAE) Buffer elution : ionic strength or pH change Cation Exchanger

Affinity Chromatography Purification of the glucose binding protein concanavalin A by passing through a glucose-coated resin Antibody purification by passing through an antigen-coated resin Buffer elution via increasing ionic strength or ligand displacement

Protein Monitoring UV absorbance (real time) Bradford reagent (fractions) Coomassie blue or silver staining (gel analysis) Proteins migrate large to small from top to bottom of gel; a molecular ladder calibrates position with protein size.

Polyacylamide-Gel Electrophoresis SDS denatures and negatively coats the proteins for analysis Separation based on size

Isoelectric Focusing Chromatography pH gradient gel in which proteins migrate to their isoelectric point; separation based on protein charge Migration Separation

Two-Dimensional Gel Electrophoresis Resolution initially by charge (isoelectric focusing) and then attached to SDS-PAGE for molecular weight separation. Over 1,000 different proteins can be resolved from a single experiment.

Quantification of Purification Protocol

Protein Purification Electrophoretic Analysis Total protein loaded in each lane is constant

Protein Primary Sequence Protein analysis road map: Bioassay design Isolation/purification Analysis Sequencing

Protein Sequencing by Edman Degradation Labeled amino-terminal residues are sequentially removed and chromatographically identified Is it necessary that a polypeptide is purified to homogeneity for this analysis?

Labeling Reaction for Edman Degradation

Amino Acid Identification by Retention Time Standardization PTH-Gly Why is there a limit on the number of cycles that can be performed?

Sequenced Polypeptide Fragments are Connected by Aligning Overlapping Sections

Polypeptide Site-Specific Cleavage Tools

Secondary Structure Predictions by Computer Modeling

Sickled Red Blood Cells in Capillaries Single amino acid substitution at position 6 in β-chain (Val → Glu) Decreased solubility of deoxyhemoglobin therefore more likely clumping in capillaries Symptoms include anemia (greater blood turnover), stroke, and bacterial infections (poor circulation)