Gel Filtration Chromatography

1
Gel Filtration Chromatography

• The method mostly involves the separation of the
  proteins based on its molecular size. This method
  is also known as Size exclusion chromatography?

• 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 Gel Filtration Chromatography
  
• Level(UG/PG) UG
• 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
1

• After interacting with this learning object, the
  learner will be able to
• Define the column preparation for the
  chromatographic technique
• Prepare elution buffers for the experiments
• Analyse the mechanism behind the protein
  purification
• Assess the troubleshooting steps involved in the
  experiments

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Master Layout
1
Column Preparation (Slide 5-13)
2
Sample addition (Slide 14-16)
Elution (Slide 17-21)
3
UV-visible spectrometry (Slide 22-24)
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Definitions and Keywords
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1. Gel filtration chromatography The protein
separation is based on the molecular size of the
protein, gel packing in the column and the
molecular filtration efficiency of the gel 2.
Size exclusion beads The manufactured beads that
has pore size depending on the molecular
weight/size of the protein to be purified. These
beads act as stationary phase 3. Elution buffer
Elution buffer consists of, 0.1 SDS, 50mM TRIS
that can be used as the mobile phase to elute out
the protein from the column
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Step 1
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T1Column Preparation
2
Beaker
Magnetic bead
3
Magnetic stirrer
Description of the action
Audio Narration (if any)?
Magnetic stirrer instrument helps for evenly
distribution of solute into the solvents
at faster rate.
Show magnetic stirrer instrument. Let user place
the beaker on it. Display the beaker containing
powder at bottom, liquid layer on top and a
magnetic bead at the bottom. Instruct user to ON
the instrument, let user control the speed nob
and regulate it accordingly to control the mixing
speed in the beaker. Animate powder getting into
the solution. Show a turbid solution turning
colorless
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5
6
Step 1
1
T1Column Preparation
2
Measuring balance
3
Audio Narration (if any)?
Description of the action
Show a measuring balance, with display, ON, OFF
and TARE/0 buttons on it. let user ON it, display
reading as 0.000g, let user picks up the paper
from the rack, makes 1/10 of folding on the sides
and places it on the balance. Now the display
reading changes to 0.003g. Instruct user to TARE
the reading. And animate to click the tare
button. Once user clicks it, reading must show
0
When measuing with paper, the weight of the paper
need to be tared from actual reading.
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7
Step 2
1
T1Column Preparation
Tris Base
SDS
2
Audio Narration
Description of the action
3
Prepare Elution buffer consists of, 0.1 SDS,
50mM TRIS, which is used during the equilibration
step.
Let user pick up SDS, tris base, measuring
cylinder from the rack and keeps it on the table
next to balance. Instruct user to weigh 0.1g of
SDS, let user tare the balance, user should click
on the SDS bottle, uncap it, with help of spatula
weigh the required amount on a paper over the
balance. Display a gradual increase in reading
with quantity addition. if the gram exceeds user
should remove some quantity or if it less add the
quantity to get the exact required amount. After
weighing transfer the quantity to beaker. Now
weigh 0.12g accordingly for tris base.
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8
Step 3
1
T1Column Preparation
2
Audio Narration
Description of the action
3
Measuring cylinder helps in making up the final
required volume.
Now instruct the user to take water bottle, open
the cap, take 100ml measuring cylinder, measure
90 ml. Let user remove the excess water if level
crosses 90ml mark. Transfer it to beaker. Now
take the beaker, shake it to make a proper mix as
shown in slide 10. Animate the powder getting
into the solution. Now set the pH to 8.5 by
using pH meter.
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Step 4
1
T1Column Preparation
2
STD 1
STD 2
3
Audio Narration
Description of the action
Display standard pH bottles and pH instrument and
deionized water, discard bottle placed on a
table. Instruct user to caliberate the
instrument. Let user ON the instrument. Initially
for the pH rod is dipped in in dipped in 3M KCl.
Now show like user taking out the rod and washing
it with deionized-water, let user cleans the rod
with tissue. Now pick the STD 1 , uncap it, dip
the cleaned rod into the solution, user must
click read button with display showing 7. Now
clean the rod and repeat the step to note down
the reading for STD 2 and now the display should
show 10
Before the pH reading, pH instrument need to be
calibrated with standards. Once with STD 1 at pH
7 and with STD 2 at pH 10.

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10
Step 5
1
T1Column Preparation
2
NaOH
HCl
3
Audio Narration
Description of the action
Instruct user to set the pH for labeling buffer
pH at 8.5. Now take the Elution buffer bottle,
uncap it, dip the cleaned pH rod into the
solution. User need to click on read button.
Initially display must show a reading 6. now
instruct user to add NaOH to adjust the pH. Now
allow the user to click on NaOH bottle so that
drops of NaOH should be added with filler, user
need to mix the solution with glass rod, click on
read button and the reading should anywhere near
11. let user keeps adding the NaOH drop till the
pH display shows 8.5 and later transfer the
beaker solution to 100ml measuring cylinder to
makeup the volume to 100ml by clicking on water
and adding it to that. All action should happen
when the user clicks the hand image.
Prepare elution buffer of pH 8.5.
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Step 6
1
T1Column Preparation
Gel filtration Beads
2
Audio Narration
Description of the action
3
Let user pick up Gel filtration beads, measuring
cylinder from the rack and keeps it on the table
next to balance. Instruct user to weigh 25g of
beads, let user tare the balance, user should
click on the SDS bottle, uncap it, with help of
spatula weigh the required amount on a paper over
the balance. Display a gradual increase in
reading with quantity addition. if the gram
exceeds user should remove some quantity or if it
is less add the quantity to get the exact
required amount. After weighing transfer the
quantity to beaker. Instruct the user to click
on the bottle labeled as elution buffer and allow
him to pour to the beaker with the weighed beads
and allow it to stand for 30 minutes and animate
a clock
Prepare size exclusion column using the beads
with definite pore size which can separate
protein based on the molecular weight.
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Step 7
1
T1Column Preparation
Coulmn
DEAE
Column 1
2
3
Let user take out the empty column from the rack,
fix the stopper to close. Animate like the user
taking a beaker with beads and user click, should
pour the beads into the column and once poured
show the column as in figure.
The bead volume should be100 times than that of
the sample to be loaded.
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Step 8
1
T1Column Preparation
Buffer
Buffer
2
Column
stopper
3
Animate like the user tightening the knob at the
bottom in the column. The user must click on the
beaker labeled as elution buffer and animate like
the user pouring the solution inside the tube.
Now instruct the user to click on hands to place
a beakers at the bottom of the columns and open
the stopper . Animate like the liquid comes out
of the tube in drop to the beaker and show like
closing the stopper and show a liquid layer at
the top of the column
Equilibrate the column using elution buffer.
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Step 9
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T2 Sample addition
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sample
3
stopper
Show a tube labeled as sample and the user
should take the pipette set to 250ul, pipette out
the sample and add to the column as shown in
figure Events must happen as and when user
clicks on the pipette, animate a clock for 10
minutes
Load the sample to the gel column to carry out
filtration process.
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Step 10
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T2 Sample addition
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Column 1
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Step 10
1
T2 Sample addition
The separation is based on the molecular weight
of the sample, higher molecular weight proteins
will be washed first while the proteins of lower
molecular weight moves slower and takes time to
elute out as it passes through the pores of the
column.
2
Animate like rings of different color with some
small and large circles. the larger circle rings
must move at faster rate while the small circle
rings must move slowly by passing through the
column. Please re-draw the previous figure.
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Step 11
1
T3 Gel filtration Elution
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Elution buffer
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Step 11
1
T3 Gel filtration Elution
Now instruct the user to take the pipette set
1000ul and take the elution buffer and add to the
column show the increase in the volume in the
column and the large/small circle movement as
described in slide 20. Events must happen when
the user clicks on it
Pour the elution buffer to elute out the proteins
at faster rate.
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Step 12
1
T3 Gel filtration Elution
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20
Step 12
)?
1
T3 Gel filtration Elution
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Step 13
1
T3 Gel filtration Elution
Audio Narration
2
Show the collection tubes in a row and the
solution dropping into it. Show the tube1 with
only solution, tube2 with some large rings, user
should click on it and a tab should appear
labeled as high molecular weight proteins and
tube3 with more of small rings tube4 with some
small rings user should click on it and a tab
should appear labeled as Low molecular weight
proteins and decreased amount of small rings in
tube5,6 and less of small size rings in tube7,8
and only solution in tube9
The larger sized molecules tend to pass through
the openings between the beads, while the small
molecules pass through the gel beads taking a lot
of time to elute out.
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Step 14
1
T4 UV-visible spectrometry
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Cuvette
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5
Please re-draw the figure to show Blue and green
beads of different sizes
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Step 14
1
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 and take a cuvette as in figure and
click on phosphate buffer to take it into cuvette
and animate like keeping it inside the UV Visible
spectrometry and press auto zero Display a
value on the system as 0.000 animate like the
user opening the lid and taking out the cuvette
and discarding the solution, now animate like the
user taking the sample 1 and adding to the
cuvette, keeping it inside, closing the lid and
press absorbance show the values as in next slide
for each sample follow the same for (2-9)
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 14
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 22-24
Slide 5-13
Slide 14-16
Slide 17-21
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage

• Animation area

Interactivity area
Slide 13 Let the user remove the buffer
entirely from the column, show like the column
drying. Instruction Instruct the user to add
the buffer to the column and close the stopper,
show like some buffer still remains in the top of
the column .
Instructions/ Working area
Credits
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Questionnaire
APPENDIX 1

• Question 1
• In gel filtration chromatography separation is
  based on
• Molecular size
• Molecular structure
• Confirmation
• Stereochemistry
• Question 2
• Gel filtration chromatography also known as
• Size exclusion chromatography
• Ion exchange chromatography
• Affinity chromatography
• Chromatofocussing
• Question 3

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Questionnaire
APPENDIX 1

• Question 4
• In gel Filtration chromatography stationary phase
  is
• Buffer containing acid
• Buffer containing Base
• Buffer Containing salt
• Size exclusion beads
• Question 5
• Volume of sample to be loaded in the column is
• Sample volume100column size
• Column size100sample volume
• Samplevolumecolumn size/100
• Sample volume column size

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APPENDIX 2
Links for further reading

• Reference websites
• http//www.mnstate.edu/provost/sizeexclusionprotoc
  ol.pdf
• http//kirschner.med.harvard.edu/files/protocols/G
  E_gelfiltration.pdf

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APPENDIX 3
Summary
Size exclusion chromatography involves separation
based on molecular size of the protein. The size
of the gel beads used in the column preparation
plays a very important role in separation of
molecules.