This document discusses different chromatography techniques used to separate proteins and molecules, including: 1) Ion-exchange chromatography uses resins with attached ions that provide a positive or negative charge to separate molecules based on their charge. 2) Gel-filtration chromatography can be used to separate proteins based on their size and molecular weight. 3) Affinity chromatography separates molecules based on their ability to bind specifically to antibodies or other ligands attached to resins. 4) Fast-performance liquid chromatography is commonly used and pumps samples through columns for high-resolution separation of proteins and other molecules.

1. Biotechnology: Science for the New Millennium Lab 9c Using Ion Exchange Chromatography to Separate Proteins Ellyn Daugherty SMBCP, San Mateo CA www.BiotechEd.com www.SMBiotech.com www.emcp.com/biotech www.sargentwelch.com/biotech

2. The Amylase Project Model of rDNA/protein Business < Chapters 1-5 Basic SLOP < Lab 5f Amylase PAGE (revised) < Lab 6e Amylase Producing Bacteria < Lab 6c Amylase Activity Assay < Lab 6d Amylase ELISA and W Blot (new) < Labs 7a/7b/7f/7g Amylase Spectrophotometry < Lab 8b Restriction Digestion of pAmylase < Lab 4j/8b/8g DNA Gel Electrophoresis (revised) < Lab 8c pAmy Transformation of E. coli < Lab 9c/9d Amy Ion-Ex Chromatography < Lab 8g/4h Genomic & plasmid DNA Isolation < Lab 13i Amylase Gene PCR (new)

3. Using Chromatography to Study and Separate Molecules Paper Chromatography Paper chromatography. Molecules separate as they move up the paper. The distance that the molecules travel depends on their size and solubility in the solvent.

4. Thin-Layer Chromatography Thin-layer chromatography. Molecules separate as they move through the silica gel. Thin-layer chromatography is used to separate small molecules, such as amino acids. Using Chromatography to Study and Separate Molecules

5. Column Chromatography How a Column Works

6. Column Chromatography Open or Gravity Column

7. Column Chromatography Gel-Filtration (Size-Exclusion) Chromatography Gel Filtration Resin. When starting protein purification, technicians sometimes use a gel-filtration (size-exclusion) column first. They know the molecular weight of their protein, so they can often eliminate several contaminant proteins by a quick run through a sizing column.

8. Ion-Exchange Chromatography Ion Exchange Resin. Resins are manufactured with ions attached. The ions present a certain degree of positive or negative charge, depending on the buffer pH.

9. Affinity Chromatography Affinity Chromatography. Separating molecules based on shape is often done using antibody resin. Antibodies recognize only certain antigens and will bind those and pull them out of solution (fraction #3).

10. Fast-Performance Liquid Chromatography (FPLC) Fast-Performance Liquid Chromatography. Pumps push the buffer or sample through tubing, into and through the column. As fractions come off the column, they are run through a spectrophotometer that determines the protein concentration of the sample. Column Chromatography in Biomanufacturing

11. Column Chromatography in Biomanufacturing Fast-Performance Liquid Chromatography (FPLC)

12. High-Performance Liquid Chromatography (HPLC) Greatly improved ability to separate, purify, identify, and qualify samples.

13. Biotechnology Curricular Support www.emcp.com/biotech for books/IRC help Cheryl Monson [email_address] www.BiotechEd.com for PPTS, syllabi, activities, hints, links, etc [email_address] www.SargentWelch.com/biotech for materials/equipment support Amy Naum [email_address]