Biotechnology: Science for the New Millennium Lab 9c

1
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 www.emcp.co
m/biotech www.sargentwelch.com/biotech
2
The Amylase Project Model of rDNA/protein
Business
lt Chapters 1-5 Basic SLOP lt Lab 5f Amylase PAGE
(revised) lt Lab 6e Amylase Producing Bacteria lt
Lab 6c Amylase Activity Assay lt Lab 6d Amylase
ELISA and W Blot (new) lt Labs 7a/7b/7f/7g
Amylase Spectrophotometry lt Lab 8b Restriction
Digestion of pAmylase lt Lab 4j/8b/8g DNA Gel
Electrophoresis (revised) lt Lab 8c pAmy
Transformation of E. coli lt Lab 9c/9d Amy Ion-Ex
Chromatography lt Lab 8g/4h Genomic plasmid DNA
Isolation lt 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
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.
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
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.
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.BiotechEd.com for PPTS, Syllabi,
Acitivities, Hints, Links, etc Ellyn_at_BiotechEd.com
www.emcp.com/biotech Cheryl Monson
CMonson_at_emcp.com for books/IRC help
www.SargentWelch.com/biotech Amy Naum for
materials/equipment support Amy_Naum_at_vwreducation
.com