Transcript Document

Using Ion Exchange Chromatography
to Separate Proteins
Biotechnology: Science for the New Millennium Lab 9c
Ellyn Daugherty
SMBCP, San Mateo CA
www.BiotechEd.com
www.SMBiotech.com
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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)
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 Resin.
Resins are manufactured
with ions attached. The ions
present a certain degree of
positive or negative charge,
depending on the buffer pH.
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).
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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