Transcript Affinity Chromatography Ion Exchange

Size Exclusion Chromatography
Proteins
• 75% of dry matter in living
things is protein.
• Biologist must purify protein
from other proteins in the cell.
• What are characteristics of
Proteins we could use to
separate them?
Characteristics of Proteins
• Hydrophilic
• Hydrophobic
• Positive
• Negative
• Size
• Active site
Amino Acids
Hydrophilic
Hydrophobic
Size
Active Site
• Biological
function
• Individual
chemical
structure
Chromatography
• Allows the separations of
individual component from
complex mixtures
• Biology
– Separation of chlorophyll
• Biotechnology
–Purify biological molecules
Separation of Chlorophyll
Protein Purification
• Affinity Chromatography
• Ion Exchange
–Anion exchange
–Cation exchange
• Gel Filtration Chromatography
–size exclusion chromatography
Affinity Chromatography
• Biological
function
• Individual
chemical
structure
Antibody isotypes of mammals
Name
Types
Description
IgA
2
IgD
1
IgE
IgG
IgM
Found in mucosal areas, such as the gut,
respiratory tract and urogenital tract, and
prevents colonization by pathogens.[10] Also
found in saliva, tears, and breast milk.
Functions mainly as an antigen receptor on B
cells that have not been exposed to
antigens.[11] Its function is less defined than
other isotypes.
1
Binds to allergens and triggers histamine
release from mast cells and basophils, and is
involved in allergy. Also protects against
parasitic worms.[6]
4
In its four forms, provides the majority of
antibody-based immunity against invading
pathogens.[6] The only antibody capable of
crossing the placenta to give passive immunity
to fetus.
1
Expressed on the surface of B cells and in a
secreted form with very high avidity. Eliminates
pathogens in the early stages of B cell
mediated (humoral) immunity before there is
sufficient IgG.[6][11]
Antib
ody
Com
plex
es
1. Affinity Chromatography
• Loading
affinity
column.
2. Affinity Chromatography
• Proteins sieve
through matrix
of affinity beads.
3. Affinity Chromatography
• Proteins
interact
with
affinity
ligand with
some
binding
loosely
and others
tightly.
4. Affinity Chromatography
• Wash
off
proteins
that do
not
bind.
5. Affinity Chromatography
• Wash off
proteins
that bind
loosely.
6. Affinity Chromatography
• Elute
proteins
that
bind
tightly
to ligand
and
collect
purified
protein
of
interest.
Affinity Chromatography Animation
Animation
Affinity Chromatography
Ion Exchange Chromatography
• separates biomolecules based
on differences in their
–anionic
–cationic
• charge characteristics
Ion Exchange Chromatography
• Anion
exchange
• Cation
exchange
Gel Filtration Chromatography
Gel Filtration Chromatography Animation
Animation
Hemoglobin
• Brown
• 65,000 Daltons
• 4 sub units
Hemoglobin
Vitamin B12
• Pink
• 1350
Daltons
Vitamin B12
1. Obtain 12 collection tubes and label
ten sequentially from 1 to 10.
2. Label the final two tubes “Waste” and
“Column Buffer”.
3. Using a clean pipette, transfer 4 ml of
column buffer into the tube labeled
“Column Buffer”.
Column Bed
Vocabulary
• Buffer
–Liquid used to
dissolve
biomolecules
• Fractions
–Samples
collected
4. Remove the cap and snap
off the end of the sizing
column. Allow all of the
buffer to drain into the
waste tube.
5. Cap the bottom of the
column.
6. Place the column onto
tube 1.
7. You are now ready to
load the protein sample
onto the column.
8. Uncap the column.
**It is important to uncap the
column only when you are
ready to load your protein—
you do not want your column
to run dry.
9. Using a pipette, add one
drop of protein mix onto the
top of the column bed.
**The pipette should be
inserted into the column and
the drop should be loaded
just above the top of the
column so that it minimally
disturbs the column bed.
10. As soon as the drop of
protein mix enters the
column bed, add 250 μl
of column buffer to the
top of the column.
11. Let the buffer run
down the side of the
tube and onto the top
of the bed.
12. Begin to collect drops
into tube 1.
13. Add another
250 μl of
column
buffer to the
top of the
column.
14. Continue to
collect drops
into tube 1.
15. Add 3 ml of column buffer to the top of
the column matrix.
16. Transfer the column to tube 2 and
count the drops that enter into each
tube.
17. Collect 5 drops of buffer into tube 2.
• Hemogloblin
moves faster
through the
column than the
Vitamin B-12
18. When 5 drops have been collected into
tube 2, transfer the column onto tube
3.
19. Collect 5 drops of buffer into each
tube.
20. When 5 drops have been collected into
a tube, lift it off and transfer it to the
next tube.
21.Continue collecting 5 drops into each tube.
22.When you reach tube 10, collect a total of 10
drops.
23.Sketch your results.
Results