Transcript appendix 1

Immunoprecipitation
The experiment involves the precipitation of protein of interest using the
specific antibody against the protein followed by addition of Protein A tagged
beads that can aid in the precipitation . The method is mostly specific to purify
our protein of interest
Related LOs: Antibody reaction,
> Prior Viewing – IDD-1. Extraction of bacterial protein, IDD-11. Protein
quantification
> Future Viewing – IDD-17. SDS-PAGE, IDD-33. Western blot assay, IDD-38. Stable
isotope labeling using amino acids in cell culture (SILAC)

Course Name: Immunoprecipitation
 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-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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Define the immunoprecipitation of the protein
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Carry out separation step for precipitated protein
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Infer the antibody binding properties
4.
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Assess the troubleshooting steps involved in the
experiments.
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Master Layout
Protein Precipitation Slide 510
Protein Soubilization Slide 11
Antibody addition and
incubation
Addition of protein –A tagged
beads
Slide
15-19
Storage
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Slide
12-14
Slide 20
Please animate the figures for layout from each step.
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Definitions and Keywords
1. Antibody: are nothing but proteins that are raised against the particular
protein which has higher specificity towards that protein
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2. Sepharose beads: Polysaccharide polymer from sea weeds that can be
used to immobilize protein by binding to cysteine residues in the protein.
3. Lysis Buffer: A cocktail of reagents used for cell lyses.
a) Trichloro acetic acid: The acid used in the lyses buffer for lyses of the
cell and helps to precipitate the protein.
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b) Acetone: One of the constituent of lyses buffer used to denature the
protein.
c) Dithiothreitol: Constituent of lyses buffer used to reduce the disulfide
bonds in the protein
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Step 1:
T1: Protein Precipitation
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Description of the action
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Instruct user to go through the or
glance in few slides the IDD-1.
Extraction of bacterial protein.
Audio Narration
(if any)
User must be aware of protein
extraction protocol.
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Step 2:
T1: Protein Precipitation
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Description of the action
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Show a measuring balance the user
should click “ON” to on the Instrument,
pick paper from rack, fold it 1/10 on the
edges, place it on the balance so that
balance reads 0.03g and the user should
press ”0” on the balance to make the
reading to “0.00”. Animate the action,
whenever user starts to weigh any
reagents.
Audio Narration
(if any)
Clean the surface of
the balance, Tare the
weight of the paper
before weighing for
each reagent.
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Step 3:
T1: Protein Precipitation
NaOH
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Description of the action
Instruct user to prepare 0.2M NaOH. Animator redraw
above figure as shown. Let user takes out the bottles
from the racks. Instruct user to weigh NaOH, tare the
balance like in previous slide, the user should pick the
spatula open the lid of the NaOH to weigh 0.098g
separately and put in the tube labeled as “0.2M NaOH”.
In case if the gram exceeds user should remove some
quantity or if it is low add to get required amount.
Instruct user to click on water, to take out 10ml in 25ml
cylinder to transfer to 0.2M NaOH bottle, now let user
take the bottle for a brief vortex to mix the solution.
0.2M NaOH
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Audio Narration
Prepare 0.2M NaOH, which
helps to remove excess
acetone and Trichloroacetic
acid.
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Description of the action
Instruct user to take out sample from -20’C
frezzer. Let user keep it on ice, thaw the
sample tube if required. Instruct user to add
TCA-Acetone to the sample tube. Let user
ser the pipette to 1000ul and draw “TCAacetone” and pipette out into the sample
tube. show the solution slowly turning to
white turbid. Instruct the user to keep the
tube in the -20C freezer by opening it and
closing it and show a clock for 1hr
TCA-Acetone
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T1: Protein Precipitation
Sample
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Step 4:
Audio Narration
Precipitate the protein from the
rehydration buffer using 5X TCAAcetone and incubate at -20C for
1hr.
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Step 5:
T1: Protein Precipitation
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Description of the action/
interactivity
After 1hr, instruct user to open door of freezer, take out the
tubes, transfer it to centrifuge. Zoom in the rotor, balance
equal number of tubes inside the rotor of centrifuge . Close
the lid of rotor and of centrifuge with hand action. Instruct
user to set the 14000rpm, 4’ C temperature and 30minutes
time along with display. User can increase and decrease
the values of set parameters. Animate the white substance
in the tube getting settled at the bottom of the tube during
centrifuge. Kindly redraw the figures after 30 minutes,
instruct the user to click open to open the rotor and remove
the tube.
Audio Narration
(if any)
Place the sample in the centrifuge,
balance with equal number of tubes
and centrifugation helps for the
phase separation.
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Audio Narration
Description of the action
Zoom the tube to show white substance at Add 100ul of 0.2M NaOH to
the bottom and the clear solution at the
remove excess acetone and
top.
Trichloroacetic acid that may
hinder in protein solubilization
Animator should instruct the user to
in PBS buffer.
discard the clear solution. To the pellet
instruct user to click on pipette, set to
100ul and pipette out 100ul of 0.2M NaOH
and add to the pellet
Event must happen when the user clicks
on the pipette.
Show like closing the tube and mixing.
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0.2M
NaOH
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T1: Protein Precipitation
Sample
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Step 6:
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T2: Protein Solubilization
Description of the action
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Animator should show a bottle
labeled as “PBS pH 7”
Now instruct the user to click on
pipette and set to 600ul, take the
PBS and add to the sample tube
and close the lid and show like
mixing it
Phosphate
buffer
Saline
Sample
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Step 7:
Audio Narration
Add Phosphate buffer Saline (pH 7)
to the sample for solubilization and
suspension. Now the sample is
ready for antibody reaction.
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Description of the action
Monoclonal
antibody
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T3:Antibody addition and incubation
Sample
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Step 8:
Audio Narration
Instruct the user to click on the pipette, set to 200ul Take 200ul of the sample that
and take the sample, open the lid and pipette out
corresponds to 500ug of proteins
200ul to the new tube labeled as “for
and 100ul of monoclonal antibody
immunoprecipitation”.
to the sample. For the reaction to
proceed keep the tube on rocker.
Instruct the animator to open the freezer to take a
tube labeled as “Monoclonal antibody” from the
freezer and show place it on ice. Show the change
of phase from solid to liquid (inside the tube) as the
time progresses.
Instruct the user to click on the pipette, set to 100ul
to take out “Monoclonal antibody”, open the lid and
pipette out 100ul into the tube labeled as “for
immunoprecipitation”
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Step 9:
T3:Antibody addition and incubation
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Rocker
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Description of the action/ interactivity
Instruct user to close the tube and
keep it on rocker placed in 4’C
room (draw the room labeled as”
cold room”) and show like the
user taking the cello tape to fix the
tube on the rocker.
Show a clock running for 12 hours
Audio Narration
(if any)
Place the tube at 4’C overnight
with constant movement.
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Step 11: T3:Antibody addition and incubation
Antibody
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Other Proteins
Protein of interest
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Description of the action
Redraw the figure, animate red and
blue circles, the user should click on red
circle to know about it and draw a Y
shaped (red) structure as shown .
Animate like the Y shaped structure
slowly moving down and getting bind to
the red circle.
Audio Narration
Due to the specificity of the
monoclonal antibody, it binds to the
protein of interest.
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Step 12: T4:Addition of protein –A tagged beads
Description of the action
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Show the tube labeled as “ sephradose bead protein –G
coupled”. Instruct the user to take the sephradose beads
and weigh the beads (100mg) as in slide-7.
Animate like the user taking the pipette set to 1000ul ,
take PBS and add to the beads tube. Animate a clock
running for 1hour then show a centrifugation step as in
slide-9 but set the time for 10mins. After 10 minutes
show like the user opening the lid and taking out the
tube, zoom in to show two layers, and show like user
removing the liquid part with pipette for discard. now
instruct the user to add 200ul of PBS-BSA solution by
setting the pipette.
Again show a centrifugation step as shown earlier,
remove the liquid part and instruct the user to set the
pipette to 400ul to add PBS to the same tube. Carry out
centrifugation step followed by removing the
supernatant. The events should happen with user
interaction. Animate the formation of small beads in the
tube.
Audio Narration
Prepare the Protein G
coupled sephradose beads
by pretreating it with the
0.1%w/v PBS-BSA and PBS
solution.
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Step 13: T4:Addition of protein –A tagged beads
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Description of the action
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Animate like the user picking the tip
and the scissors in the hand. Ask
user to click on the scissors to cut the
tip at the end, show a blunt end tip.
Audio Narration
Due to the sharp opening of the tip, it
wont be able to draw the beads. For this
reason the tip need to me cut to have a
larger opening for the beads.
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Step 14: T4:Addition of protein –A tagged beads
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Description of the action
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Instruct the user to take the pipette and
load it with the blunt end tip (cut in
previous slide) and set the pipette to 700ul
and take the beads inside it and add to the
tube labeled as “ For
immunoprecipitation”(from slide 12)
Events must happen when the user clicks
on the pipette
Audio Narration
Take 700ul of the beads and add to the
sample for precipitation reaction.
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Step 15: T4:Addition of protein –A tagged beads
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Protein-G
coupled bead
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Step 16: T4:Addition of protein –A tagged beads
Protein-G
coupled
bead
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Description of the action
Redraw the figure, animate red and blue circles, the user
should click on green circle to know about it and draw a
circle/rectangle shaped (green) structure as shown .
Animate like circle/rectangle shaped structure slowly
moving down and bind to the red Y shaped geometry.
Show like keeping it in the rocker as shown in slide:14
and centrifugation in slide:9 and time only for 10 minutes.
Instruct the user to remove the liquid part using the
pipette and zoom in to show the white substance at
bottom.
Audio Narration
Protein –G coupled protein
binds to antibodies and
settle down on
centrifugation. The settled
down material can be
taken for further
processing.
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Step 17: T5:Storage
Description of the action
Show a tube labeled as “loading
buffer” in blue color
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Instruct the user to set the pipette
to 50ul and take out the loading
buffer and add to the white
substance. The tube can now be
stored at -20’C freezer for further
analysis of the sample.
Audio Narration
After precipitation process the
sample can be solubalized into
loading buffer. For more
information please refer the IDD17. SDS-PAGE and IDD-33.
Western blot assay.
Slide 510
Tab 01
Slide 11
Tab 02
Slide
12-14
Tab 03
Slide
15-19
Slide 20
Tab 04
Tab 05
Tab 06
Tab 07
Name of the section/stage
Animation area
INTERACTION 1: slide-19: user going for longer
centrifugation time of 20min and proceeds further.
Interactivity
area
Instructions: along with protein bound antibodies lot other
unnecessary proteins get settled so user need to set the
specified centrifugation time 14000rpm for 10min.
Button 01
INTERACTION 2: slide-12: let user calculate the antibody
labeling depending upon the protein concentration.
Button 03
Button 02
Instructions: the ratio for protein with antibody must be
2:1.
Instructions/ Working area
Credits
APPENDIX 1
Questionnaire:
Question 1:
Immuno precipitation means
a) Precipitating antibodies
b) Precipitating cells
c) Precipitating the protein of interest using monoclonal antibodies
d) Precipitating the antigens
Question 2:
What kind of beads are used for the precipitation
a) Sepharose
b) Sucrose
c) Glass
d) Glucose
Question 3:
Sephraose is tagged with
a) Protein A
b) Protein B
c) Protein Z
d) Antibody
APPENDIX 1
Questionnaire:
Which of the reagent is used for neutralizing Trichloroacetic acid?
a) Nacl
b) NaOH
c) Na2CO3
d) NaHCO3
Geometry of antibody is--------------- shape
a) Y
b) X
c) W
d) B
APPENDIX 2
Links for further reading
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Reference websites:
http://www.abcam.com/ps/pdf/protocols/Immunoprecipi
tation%20protocol%20(IP).pdf
Laura Trinkle-Mulcahy et,al., Identifying specifi c
protein interaction partners using quantitative mass
spectrometry and bead proteomes, JCB article,
October 20, 2008
APPENDIX 3
Summary
The method mostly involves protein precipitation followed by immunoprecipitation of
the protein using the antibodies followed by addition of sepharose beads with protein –
A to precipitation