Transcript Affinity Chromatography

Affinity Chromatography
Affinity chromatography is based on the principle of
specific interaction between the protein or antigen
and antibody for separation of biomolecules
Related LOs: Column preparation, Chromatographic technique
> Prior Viewing – IDD-6. Extraction of serum protein, IDD-42. Liquid chromatography
- affinity chromatography
> Future Viewing – IDD-38. Stable isotope labeling using amino acids in cell culture
(SILAC), IDD-37. Isotope-coded affinity tags (ICAT), IDD-39. LC-MSMS data analysis
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Course Name: Affinity Chromatography
 Level(UG/PG): PG
 Author(s): Dinesh Raghu, Vinayak Pachapur
 Mentor: Dr. Sanjeeva Srivastava
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*The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license
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Learning objectives
After interacting with this learning object, the learner will
be able to:
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Prepare elution buffers required for experiment
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Analyse the mechanism behind the protein purification
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Define the column preparation for the chromatographic
technique
Assess the troubleshooting steps involved in the
experiments.
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Master Layout
Column Preparation Slide 5-6
Slide 7-8
Sample loading
Sample separation
Slide 9-14
Slide 15-17
UV-visible spectrometry
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Definitions and Keywords
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Affinity purification: A chromatographic purification procedure that makes
use of specific interactions between the analyte of interest and the capture
analyte immobilized on the column. In some application Ni-His, iron,zinc,
gallium column are used for analysis and in ICAT, avidin affinity
chromatography is employed due to its specificity of interaction with biotin.
Isotope Coded Affinity Tagging (ICAT): ICAT is an in vitro labeling
technique that modifies peptides or proteins specifically at the cysteine amino
acid residue and can be used for accurate quantitation of protein expression.
Light ICAT label: The light ICAT reagent consists of a Cys-reactive group,
an ICAT linker consisting of hydrogen atoms and a biotin tag. The chemically
reactive group forms covalent bonds with peptides or proteins while the
affinity tag enables the protein to be isolated by affinity chromatography in a
single step that is used to tag the control sample.
Heavy ICAT label: The heavy ICAT label consists of a Cys-reactive group,
an ICAT linker consisting of heavy deuterium isotope and a biotin tag that is
used to tag the Drug treated sample.
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Step 1:
T1:Column Preparation
Coulmn
AVIDIN-Column
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Description of the action
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Instruct the user to go through the IDD-37.
Isotope-coded affinity tags (ICAT) slide from 31
to 47.
Audio Narration
The samples for
analysis is labeled
with biotin tag.
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Step 2:
T1:Column Preparation
Coulmn
AVIDIN-Column
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stopper
Description of the action
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Animate like the user taking a column from the
fridge, by opening it and removing the cover and
tightening the stopper at the bottom in the
column. Zoom in to show the column as above
Audio Narration
Take the avidin
column from the
fridge to carry out
pre-treatment of
the column.
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Step 3:
T2:Sample loading
water
Distilled
water
Column
stopper
Description of the action
Animate like the user tightening the stopper at the bottom
in the column. The user must click on the beaker labeled
as “distilled water” and animate like the user pouring the
water inside the tube using the pipette by setting full
volume. Now instruct the user to place a beaker at the
bottom of the columns and open the stopper. Animate like
the liquid coming out of the tube like drops into the beaker
and show like closing the stopper.
Audio Narration
Wash the column using
distilled water to remove the
buffer material used in
column storage.
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Step 4
T2:Sample loading
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sample
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stopper
Description of the action
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Show a tube labeled as “tube 5,6” from slide: 56-57 of
Load the sample to the avidin
IDD-37. Isotope-coded affinity tags (ICAT) and user
column.
should take the pipette set to 100ul, pipette out the
sample and add to the column as shown in figure. Repeat
the step until all the samples are in the column.
Events must happen as and when the user clicks on the
pipette animate a clock for 10 minutes
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Audio Narration
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Step 5:
T3:Sample separation
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Avidin
labeled
beads
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Column 1
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Step 5:
T3:Sample separation
Light (L)
Heavy (H)
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Description of the action
Animate like rings of different color
labeled as Light (L) and similarly other
rings of different colors labeled as Heavy
(H) show like the L and H binding to the
column while rings without H or L
labeling comes out. Please re-draw the
above figure.
Audio Narration
The separation is based on the
affinity of the biotin in the tag to the
avidin in the column, the protein
which have taken up heavy and light
isotopes tend to show affinity to the
column.
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Step 6:
T3:Sample separation
Description of the action
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Now instruct the user to take
the pipette set 1000ul and take
bottle labeled as 0.1% formic
acid to add into the column.
show the increase in the
volume in the column and the
circles with different labeling
moving. show movement as
described in slide:9 and 10.
Events must happen when the
user clicks on it
Audio Narration
Pour 0.1% formic acid containing
0.1ml formic acid and 99.9 ml water
to the column, to elute the bound
molecules.
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Step 6:
T3:Sample separation
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Step 6:
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T3:Sample separation
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Step 7:
T3:Sample separation
Description of the action
Show the collection tubes in row and the
solution dropping into it. Show in tube:1 only the
solution, tube:2 with some unlabeled rings, user
should click on it and a tab should appear to
display ”unlabeled proteins” and tube:3 with
more of unlabeled rings, tube:4 with some
labeled rings user should click on it and a tab
should appear labeled as ”Isotope tagged
proteins” and increased amount of L and H
rings in tube 5,6 and less of L and H rings in
tube 7,8 and only solution in tube 9. Instruct the
user to take the tube 5,6,7,8 for analysis
Audio Narration
The protein that show low
interaction with the beads
will be eluted first followed
by moderately interacting
protein and highly
interacting proteins. Here
the proteins that are tagged
with biotin label show more
interaction when compared
to other proteins.
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Step 7:
T4: UV-visible spectrometry
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Cuvette
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Step 7:
T4: UV-visible spectrometry
Description of the action
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Show a instrument labeled as “UV –visible
spectrometry” and the samples in the stand as
shown in figure. Animate buttons like “start, auto
zero, absorbance, stop” on the instrument
Now instruct the user to switch on the instrument,
set the wavelength to 595nm by pressing on
numbers. Open the lid of the instrument, take a
cuvette as in figure and click on phosphate buffer
to take it into the cuvette and animate like keeping
it inside the UV Visible spectrometry. Press “auto
zero” display a value on the system as “0.000”.
animate like the user opening the lid and taking the
cuvette and discarding the solution, now animate
like the user taking the sample:1 transferring it to
the cuvette, keeping it inside, closing the lid and
press absorbance to show the values as in next
slide. For other sample (2-9) follow the same step
like above.
Audio Narration
Detect the presence of
protein of using the UV –
visible spectrometry. The
high absorbance reading
indicate the presence of
protein. For more
information on the UVVisible spectrometry
please go through IDD-50
basic instrumentation.
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Step 7:
T4: UV-visible spectrometry
Sample
absorbance
Volume of sample
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0.12
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0.229
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0.303
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0.457
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0.533
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0.681
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0.71
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0.62
0.65
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Slide 5- Slide 76
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Tab 01
Slide 914
Tab 02
Tab 03
Slide
15-17
Tab 04
Tab 05
Tab 06
Tab 07
Name of the section/stage
Interactivity
area
Animation area
Interaction 1: slide-16: user getting zero reading from
tubes 6-9.
Instructions: user need to increase the concentration of
elution buffer to remove out the bound proteins.
Button 01
Button 02
Button 03
Instructions/ Working area
Credits
APPENDIX 1
Questionnaire:
Question 1
In affinity chromatography separation is based on
a) Molecular size
b) Molecular structure
c) specificity
d) Stereochemistry
Question 2
Affinity chromatography mostly uses
a)
b)
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d)
Antibody coated beads
Column with empty beads
Only mobile phase
Only column
Question 3:
Avidin-Biotin chromatography mobile phase is
a) 0.1% formic acid
b) Buffer containing Base
c) Buffer Containing salt
d) Size exclusion beads
APPENDIX 1
Questionnaire:
Question 4:
In affinity chromatography stationary phase is
a) Buffer containing acid
b) Buffer containing Base
c) Buffer Containing salt
d) Beads with specific proteins/metal that react with tagged protein
Question 5:
biotin binds
a) Antibodies
b) Streptavidin
c) Antigen
d) column
APPENDIX 2
Links for further reading
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Reference websites:
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www.mnstate.edu/provost/sizeexclusionprotocol.pdf
2.
www.younglin.com/brochure_pdf/waters/lcGPC.pdf
APPENDIX 3
Summary
Affinity chromatography involves separation based specific interaction
between the tagged protein and packing column used. The packing
material used for the separation plays a very important role in
separation of proteins.
Steps involves: elution buffer preparation, sample loading, elution and
analysis using UV-Visible spectrometry.