Using Ion Exchange Chromatography to Separate Proteins Biotechnology

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

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

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.

Using Chromatography to Study and Separate Molecules 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.

Column Chromatography How a Column Works

Column Chromatography Open or Gravity Column

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.

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

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).

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

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

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