Affinity Chromatography

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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
• gt Prior Viewing IDD-6. Extraction of serum
  protein, IDD-42. Liquid chromatography - affinity
  chromatography
• gt 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
• Course Name Affinity Chromatography
  
• Level(UG/PG) PG
• Author(s) Dinesh Raghu, Vinayak Pachapur
• Mentor Dr. Sanjeeva Srivastava

The contents in this ppt are licensed under
Creative Commons Attribution-NonCommercial-ShareAl
ike 2.5 India license
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Learning objectives
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• After interacting with this learning object, the
  learner will be able to
• Define the column preparation for the
  chromatographic technique
• Prepare elution buffers required for experiment
• Analyse the mechanism behind the protein
  purification
• Assess the troubleshooting steps involved in the
  experiments.

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Master Layout
1
Slide 5-6
Column Preparation
2
Slide 7-8
Sample loading
Slide 9-14
Sample separation
3
Slide 15-17
UV-visible spectrometry
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5
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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
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T1Column Preparation
Coulmn
DEAE
AVIDIN-Column
2
3
Instruct the user to go through the IDD-37.
Isotope-coded affinity tags (ICAT) slide from 31
to 47.
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The samples for analysis is labeled with biotin
tag.
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Step 2
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T1Column Preparation
Coulmn
DEAE
AVIDIN-Column
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3
stopper
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
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Take the avidin column from the fridge to carry
out pre-treatment of the column.
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Step 3
1
T2Sample loading
water
2
Distilled water
Column
stopper
3
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.
Wash the column using distilled water to remove
the buffer material used in column storage.
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Step 4
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T2Sample loading
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sample
3
stopper
Show a tube labeled as tube 5,6 from slide
56-57 of IDD-37. Isotope-coded affinity tags
(ICAT) and user 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
Load the sample to the avidin column.
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Step 5
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T3Sample separation
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Avidin labeled beads
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Column 1
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Step 5
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T3Sample separation
Light (L)
Heavy (H)
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3
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.
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
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T3Sample separation
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 slide9 and 10. Events must happen
when the user clicks on it
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
1
T3Sample separation
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Step 6
)?
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T3Sample separation
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Step 7
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T3Sample separation
Audio Narration
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Show the collection tubes in row and the solution
dropping into it. Show in tube1 only the
solution, tube2 with some unlabeled rings, user
should click on it and a tab should appear to
display unlabeled proteins and tube3 with more
of unlabeled rings, tube4 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
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
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T4 UV-visible spectrometry
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Cuvette
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Step 7
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T4 UV-visible spectrometry
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 sample1 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.
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 UV-Visible spectrometry
please go through IDD-50 basic instrumentation.
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Step 7
1

T4 UV-visible spectrometry
Sample absorbance
1 0.12
2 0.229
3 0.303
4 0.457
5 0.533
6 0.681
7 0.71
8 0.62
Volume of sample
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9 0.65
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Slide 15-17
Slide 5-6
Slide 9-14
Slide 7-8
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage

• 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.

Interactivity area
Instructions/ Working area
Credits
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Questionnaire
APPENDIX 1

• Question 1
• In affinity chromatography separation is based on
• Molecular size
• Molecular structure
• specificity
• Stereochemistry
• Question 2
• Affinity chromatography mostly uses
• Antibody coated beads
• Column with empty beads
• Only mobile phase
• Only column
• Question 3

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Questionnaire
APPENDIX 1

• Question 4
• In affinity chromatography stationary phase is
• Buffer containing acid
• Buffer containing Base
• Buffer Containing salt
• Beads with specific proteins/metal that react
  with tagged protein
• Question 5
• biotin binds
• Antibodies
• Streptavidin
• Antigen
• column

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APPENDIX 2
Links for further reading

• Reference websites
• www.mnstate.edu/provost/sizeexclusionprotocol.pdf
• www.younglin.com/brochure_pdf/waters/lcGPC.pdf

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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.