1 / 22

Chapter Five Protein Purification and Characterization Techniques

Chapter Five Protein Purification and Characterization Techniques. Isolation of Proteins from Cells. Many different proteins exists within one cell • Many steps needed to extract protein of interest, and separate from many contaminants

misu
Download Presentation

Chapter Five Protein Purification and Characterization Techniques

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Chapter FiveProtein Purification and Characterization Techniques

  2. Isolation of Proteins from Cells Many different proteins exists within one cell • Many steps needed to extract protein of interest, and separate from many contaminants • Before purification begins, protein must be released from cell by homogenization

  3. How We Get Proteins Out of Cells

  4. Salting Out • After Proteins solubilized, they can be purified based on solubility (usually dependent on overall charge, ionic strength, polarity • Ammonium sulfate (NH4SO4) commonly used to “salt out” • Takes away water by interacting with it, makes protein less soluble because hydrophobic interactions among proteins increases • Different aliquots taken as function of salt concentration to get closer to desired protein sample of interest (30, 40, 50, 75% increments) • One fraction has protein of interest

  5. Differential Centrifugation • Sample is spun, after lysis, to separate unbroken cells, nuclei, other organelles and particles not soluble in buffer used • Different speeds of spin allow for particle separation

  6. Column Chromatography • Basis of Chromatography • Different compounds distribute themselves to a varying extent between different phases • Interact/distribute themselves • In different phases • 2 phases: • Stationary: samples interacts with this phase • Mobile: Flows over the stationary phase and carries along with it the sample to be separated

  7. Column Chromatography

  8. Size-Exclusion/Gel-Filtration • Separates molecules based on size. • Stationary phase composed of cross-linked gel particles. • Extent of cross-linking can be controlled to determine pore size • Smaller molecules enter the pores and are delayed in elution time. Larger molecules do not enter and elute from column before smaller ones.

  9. Size Exclusion/Gel-filtration (Cont’d)

  10. Affinity Chromatography • Uses specific binding properties of molecules/proteins • Stationary phase has a polymer that can be covalently linked to a compound called a ligand that specifically binds to protein

  11. Ion Exchange • Interaction based on overall charge (less specific than affinity) • Cation exchange • Anion exchange

  12. Electrophoresis • Electrophoresis- charged particles migrate in electric field toward opposite charge • Proteins have different mobility: • Charge • Size • Shape • Agarose used as matrix for nucleic acids • Polyacrylamide used mostly for proteins

  13. Electrophoresis (Cont’d) • Polyacrylamide has more resistance towards larger molecules than smaller • Protein is treated with detergent (SDS) sodium dodecyl sulfate • Smaller proteins move through faster (charge and shape usually similar)

  14. Isoelectric Focusing • Isolectric focusing- based on differing isoelectric pts. (pI) of proteins • Gel is prepared with pH gradient that parallels electric-field. What does this do? • Charge on the protein changes as it migrates. • When it gets to pI, has no charge and stops

  15. Primary Structure Determination How is 1˚ structure determined? • Determine which amino acids are present (amino acid analysis) • Determine the N- and C- termini of the sequence (a.a sequencing) • Determine the sequence of smaller peptide fragments (most proteins > 100 a.a) 4) Some type of cleavage into smaller units necessary

  16. Primary Structure Determination

  17. Protein Cleavage Protein cleaved at specific sites by: • Enzymes- Trypsin, Chymotrypsin • Chemical reagents- Cyanogen bromide Enzymes: Trypsin- Cleaves @ C-terminal of (+) charged side chains Chymotrypsin- Cleaves @ C-terminal of aromatics

  18. Peptide Digestion

  19. Cleavage by CnBr Cleaves @ C-terminal of INTERNAL methionines

  20. Determining Protein Sequence After cleavage, mixture of peptide fragments produced. • Can be separated by HPLC or other chromatographic techniques • Use different cleavage reagents to help in 1˚ determination

  21. Peptide Sequencing • Can be accomplished by Edman Degradation • Relatively short sequences (30-40 amino acids) can be determined quickly • So efficient, today N-/C-terminal residues usually not done by enzymatic/chemical cleavage

  22. Peptide Sequencing

More Related