Download Methodology for the extraction of Bacterial protein

Methodology for the extraction of Bacterial
protein
Extraction of the entire protein from the sample requires a optimized
protocol to increase the protein amount in the extract. The protein
extraction from the cell requires suitable reagents and technique that can
yield a better and efficient result
Related LOs: Cell culture, handling laminar air flow
> Prior Viewing – IDD:2 Plant extraction, IDD:6 Serum extraction
> Future Viewing – IDD:11 Protein Quantification, IDD: 12 Rehydration, IDD: 15 IEF,
IDD:17 SDS-PAGE, IDD:20 Staining

Course Name: Methodology for the extraction of Bacterial protein
 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:
1.
Identify the mechanism in TRIzol extraction
2.
Examine the technique involved in culture growth
3.
Interpret the results of the experiment
4.
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Assess the troubleshooting steps involved in the
experiments
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Master Layout
Inoculation of Bacterial culture (Slide 5-8)
Centrifugation followed by Buffer treatment
(Slide 9-11)
Cell Lysis by sonication (Slide 12-14)
Trizol treatment (Slide 12-14)
3
Chloroform treatment (Slide 19)
Absolute alcohol treatment (Slide 20-21)
4
Acetone precipitation (Slide 22-23)
Sample treatment with rehydration buffer
(Slide 24-27)
Store the sample at -20’C (Slide 28)
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Definitions and Keywords
1.Protein: are the biomolecules, composing of amino acid, which forms the building
block of the system and performs most of the function in the system.
2.Bacterial protein extraction: The process by the proteins from the cell are
recovered for analysis purpose is called protein extraction. The chemicals involved in
the extraction are
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a)Luria –Bertani broth (LB): The LB broth consists of yeast extract as carbon
source, peptone as amino acid source, NaCl to maintain osmo-regulation and water.
b)Trizol reagent: The reagent consists of phenol, guanidium thiocyanate and
chloroform. Phenol and chloroform helps in phase separation while guanidium
thiocyanate acts as Rnases inhibitors.
c)CHAPS: 3-{Dimethyl[3-(4-{5,9,16-trihydroxy-2,15-dimethyltetracyclo[8.7.0.02,7.011,15]
heptadecan-14-yl}pentanamido)propyl]azaniumyl}propane-1-sulfonate (“CHAPS” ) is a
zwitterioinc detergent and a constituent of rehydration buffer that is used to solubilize
the proteins including membrane proteins.
d) Urea: It is a organic compound in rehydration buffer that is used to denature
protein.
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Step 1:
T1:Inoculation of Bacterial culture
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Video File: LAMINAR air flow
Description of the action/ interactivity
Show a person sitting in front of laminar hood, zoom the
laminar working bench having , tissue roll, ethanol bottle,
burner, beaker with tooth picks. Instruct user to clean the
bench. Allow user to pick tissue, wet it with ethanol and clean
the whole working bench with user control like user should
click on the hand so that the cleaning must happen. Now
animate to close the laminar hood, click for “UV light” ON for
5min display the hood in blue color. Later after 5min, Click for
“Light” “Blower” ON buttons to start the laminar hood. Please
redraw the figures
Audio Narration
(if any)
Clean the laminar
unit thoroughly with
ethanol and expose it
to UV for further
sterilization to avoid
contamination. Later
after 5min of UV
exposure the laminar
hood is ready for
carrying out
experiment.
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Step 1:
T1:Inoculation of Bacterial culture
LB Broth
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Description of the action/ interactivity
Take out a plate labeled as master culture plate from fridge, LB
broth from the instrument labeled as incubator, the user should
click on the hand to open the incubator, take the tube and close
it and transfer it on laminar bench. With user control, open the
lid of the culture plate, show few white dots in the plates as
shown and the user should click on the hand to take a tooth
pick and pick a colony from the master culture, close the
culture plate. Take the “LB broth” tube unplug the cotton roll
with left hand. The user should click on hand to remove the
cotton plug. Now show like putting the tooth pick inside the LB
broth, and contacting the solution with the tooth pick. Please
redraw the figure.
Audio Narration
(if any)
Pick a bacterial colony
using the tooth pick
from the master
culture.
Inoculate the bacterial
colony to the sterile
broth. Always keep the
burner on during
inoculation and
perform the action
close to burner to
avoid any
contamination.
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Step 1:
T1:Inoculation of Bacterial culture
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Description of the action/ interactivity
Show the placing of inoculated tube in incubator shaker.
And the user should, open the lid, place the tube and
close the lid. Instruct user to click on settings to set the
temperature to 37’C and RPM 200 and start button so
that inside stuff in the shaker starts moving horizontally.
Please redraw the figures. Show a clock running for 8
hours
Audio Narration
Place the inoculated tube in
the shaker-incubator at 37’C
for 6-8 hours.
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Step 1:
T1:Inoculation of Bacterial culture
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Description of the action/ interactivity
Once the time is complete instruct the user
to take out the inoculated tube, open it.
zoom in to show the yellow liquid turned to
turbid. Show the transfer of grown culture
into the clean centrifuge tube as the user
clicks on it . This should happen inside the
laminar hood as shown in slide 5 Please
redraw the figure.
Audio Narration
(if any)
Transfer the bacterial culture into the
clean centrifuge tube under aseptic
condition for further processing.
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Step 2:
T2: Centrifugation followed by Buffer treatment
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5
Description of the action/ interactivity
Instruct user to open the lid of centrifuge
and drum. Zoom in the rotor, balance
equal number of tubes inside the drum.
Close the lid of rotor and of centrifuge with
hand action. Instruct user to set the rpm
(12000), temperature(4’C) and time
(10min) parameters, along with display.
User can increase and decrease the
values of set parameters. Animate the
clock for 30min. Kindly redraw the figures.
Audio Narration
(if any)
Centrifuge the culture for. 10 min at 12000
. harvest/collect
rpm maintaining at 4’C to
the culture.
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Step 2:
T2: Centrifugation followed by Buffer treatment
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Description of the action/ interactivity
After 10min, instruct user to open the lid of
centrifuge, rotor and animate the hand
action to left the tube from rotor. Now
zoom the tube with pellet on bottom and
liquid (supernatant) over it as shown in
figure. Now pipette out top liquid portion
(supernatant) completely and pipette into
empty tube, the action should take place
only when the user clicks on the pipette
and tube. Kindly redraw the figures.
Audio Narration
(if any)
Remove the supernatant completely
.
without disturbing the pellet,
now take the
pellet for further processing.
Video File: Centrifuge.MTS and Centrifuge_part2.MTSc
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Step 2:
T2: Centrifugation followed by Buffer treatment
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Description of the action/ interactivity
Show phosphate buffer bottle, instruct user
to set the pipette to 1000ul and take the
buffer in the pipette and transfer it into the
tube. The user should click on the pipette
for the action to be done.
Kindly redraw the figures
Audio Narration
(if any)
Wash the pellet with phosphate buffer
. excess broth.
thoroughly to remove the
Once broth is removed completely, cell
lysis need to be carried out.
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Step 3:
T3: Cell Lysis by sonication
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Description of the action/ interactivity
Audio Narration
(if any)
Instruct user to place the tube on ice, with cap
open. Show the sonicator instrument, place the
tube such that the tip of sonicator rod touches
the solution in the tube now the user should
click on the instrument to adjust the rod to dip
into the solution. Now display the screen of
sonicator, to make the necessary set up with
help of user interaction like setting in the
amplitude, time and pulses as in right hand side.
user should click on the sonicator to proceed
with sonication.
Keep the sample on ice and start
sonication by providing 6 cycles of
pulses for 5 sec, 20% amplitude with
5 sec gap. Sonication helps protein
extraction by cell lysis.
Video File: Sonication
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Step 3:
T3: Cell Lysis by sonication
High frequency sound
waves
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Description of the action/ interactivity
Zoom in a cell from the tube. Show the
sound waves hitting the cell and causing
cell lyses and the release of contents.
Please redraw the figure
Audio Narration
(if any)
.
High frequency sound waves
break open
the cell wall and the contents are released
into the buffer
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Step 3:
T3: Cell Lysis by sonication
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Description of the action/ interactivity
Carry out the 3 centrifuge process as
described in slide 9. The user should click
on “ Start” button for the centrifugation to
ON. Please redraw the figure.
Now show the tube containing two layers
(liquid and white substance and ask user
to set the pipette and take the liquid part to
fresh tube.
Audio Narration
(if any)
Centrifuge the contents to remove the
.
debris and collect the supernatant
for
further processing.
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Step 4:
T4: Trizol treatment
Guanidium
thiocyanate
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Phenol
Description of the action/ interactivity
The user should click on the trizol to know
the constituents of trizol reagent. please
redraw the figure
Audio Narration
(if any)
Trizol reagent consists of guanidium
thiocyanate, phenol,chloroform that
separates DNA, RNA and proteins from
each other within the solution.
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Step 4:
T4: Trizol treatment
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Description of the action/ interactivity
The user must click on the pipette, set it
1000ul and take the trizol so that the trizol
is added to the supernatant(liquid in fresh
tube). And the user should click on vortex
to start show like the content is mixed and
color should be pink. Please redraw the
figures.
Audio Narration
(if any)
Add trizol to the supernatant and vortex it
thoroughly.
Video File: Vortex
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Step 4:
T4: Trizol treatment
Chloroform
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Description of the action/ interactivity
Show the bottle labeled as chloroform and
the user should click on the bottle for
addition of chloroform to the sample when
the user clicks on the pipette, set to 200ul
and take the reagent and add to the liquid
part. Place the tube over the vortex. And
show the mixing, solution should be milky
in appearance. Please redraw the figure.
Audio Narration
(if any)
Add chloroform to the sample, mix
thoroughly till the color appear like milkshake and keep at room temperature for
5min till you get the phase separation.
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Step 4:
T4: Trizol treatment
RNA
Protein
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DNA
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Description of the action/ interactivity
Carry out centrifuge step as discussed
earlier (slide:9) after centrifuge zoom the
tube to show formation of three layers like
in figure. Please redraw the figure. User
should click on the layer to know their
constituents.
Audio Narration
(if any)
Three layers are formed after
centrifugation. The top layer containing
RNA, Middle layer with protein and the
bottom layer with DNA.
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Step 5:
T5: Absolute alcohol treatment
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Description of the action/ interactivity
Show the removal of RNA (top) layer for
discard, when the user clicks on the
pipette. Show the bottle labeled as alcohol
and the user should set the pipette to
300ul and take the alcohol in pipette and
add to the tube and mix well by inverting
the tubes with hand. The user should click
on the pipette so that the action takes
place. Later carry out centrifugation step
as in slide:9. Please redraw the figure.
Audio Narration
(if any)
Remove the aqueous layer containing RNA
without disturbing the other two layers.
RNA can be stored or discarded depending
on the requirements. Add the absolute
alcohol to the remaining layers and mix
gently till the middle layer dissolve and
keep at room temperature for 3 min.
Centrifuge the content for 5 min at 2000
rpm.
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Step 5:
T6: Acetone treatment
Supernatant
containing
Protein
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DNA
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Description of the action/ interactivity
Zoom the tube after centrifuge, to show two layers
and user click on each layer to know about it.
Show the bottle labeled as acetone and the user
should set the pipette to 1000ul and take the
acetone in pipette and add to the tube and mix well
with vortex as described earlier. Animate addition of
acetone to the sample when the user clicks on the
pipette and vortex should be done when the user
clicks on the ON button. Please redraw the figure
Audio Narration
(if any)
DNA forms pellet and the
supernatant containing protein is
recovered. Add chilled acetone to
the sample and vortex it, this
helps to precipitates the protein.
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Step 5:
T6: Acetone treatment
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Description of the action/ interactivity
Show placing of sample at -20 C for 1 hr
by opening the freezer. The user should
click freezer to open it, keep the sample
and close it. Show the clock running for an
hour. Instruct user to take out the sample
to carry out centrifuge step. centrifugation
as in slide:9. After centrifuge zoom the
tube to show white substance (pellet)
formation.
Please redraw the figures.
Audio Narration
(if any)
Place the sample at -20 C for at-least an
hour for complete precipitation. Centrifuge
the content to get white pellet to be seen
at the bottom.
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Step 6:
T7:wash buffer preparation
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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 ”0.000”
Audio Narration
(if any)
During the material measurement on
paper, the weight of the paper need to
be tarred or set to zero before
weighing.
Video File: Measuring Balance
Step 6:
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Guanidine-HCL
ethanol
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T7:wash buffer preparation
Description of the action
Let user pick up guanidine HCl with spatula, measuring
cylinder from the rack and place it on the table next to
balance. Instruct user to weigh 0.35g of guanidine let user
tare the balance, user should click on the guanidine 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 click on ethanol and the user
should pour in measuring cylinder till volume reaches 10ml
and add to the beaker containing weighed guanidine and
show like mixing well in the vortex as shown earlier.
Audio Narration
Prepare wash solution,
washing is carried to
remove excess broth and
other contaminants if
present in the solution.
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Step 6 :
T7: buffer preparation
CHAPS
water
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Rehydration
buffer (RB)
2
urea
Description of the action
Instruct user to prepare rehydration buffer (RB). Animator
redraw above figure as shown. Let user takes out the
bottles from the racks. Instruct user to weigh CHAPS and
Urea, tare the balance like in slide:22, the user should pick
the spatula open the lid of the CHAPS to weigh 0.02g and
Urea bottle to weigh 0.6g separately and put in the tube
labeled as RB. Instruct user to click on water bottle, take
out 1ml pipette, set it for 1000ul and pipette in water into
RB bottle. let user take the bottle for a brief vortex to mix
the solution.
Audio Narration
Weigh 0.6g of urea,
0.02g of CHAPS to
prepare rehydration
buffer which must be
store at 4’C.
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Step 7:
T8: Pellet washing
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Description of the action/ interactivity
Audio Narration
(if any)
Show the removal of supernatant as the user clicks on Remove the supernatant
pipette, only liquid part has to be removed ,show like carefully and air dry the pellet.
opening the tube and keeping the pellet in room
Pellet retains the pink color,
temperature show like drying and pellet drying need to carry out washing the pellet with
animated. Show the reagents which constitutes the
0.3 M guanidium –HCl in 95%
washing solution. The user should click on the
ethanol for 4 times to remove
chemical to get their role. Show like the user taking the the color and for inactivation of
pipette set to 1000ul and take the reagents and add to RNAses. Each pellet wash need
the tube And carry out centrifuge as in slide 9. Show to be followed by centrifugation
like repeating the step for 4 times. Remove the liquid by discarding the supernatant till
part in each step and add the washing solution till
pellet becomes color-less.
protein become white. Kindly redraw the figures
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Step 8:
T8: Rehydration buffer treatment
Urea
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CHAPS
Description of the action/
interactivity
Zoom-in tube showing the colorless
pellet. Instruct user to add rehydration
buffer by setting the pipette to 400ul
and take the solution and add to it.
Display the contents of rehydration
buffer when user clicks on the tube.
Kindly redraw the image
Audio Narration
(if any)
The rehydration buffer consists of CHAPS
used to solubilize the proteins including
membrane proteins. Urea used to
denature protein structure.
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Step 7:
T8: Rehydration buffer treatment
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3
4
5
Description of the action/
interactivity
Instruct user to add rehydration buffer to
sample with pipette and animate the step
accordingly. After addition close the cap of
tube and place it on rubber pad of the
vortex for proper mixing. Animate the pellet
get disappearing into the solution. kindly
redraw the images.
Audio Narration
(if any)
Add 0.4 ml of rehydration buffer (IDD-2
Extraction of serum protein, slide 8) to the dried
pellet and vortex till the pellet completely goes
into the solution.
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Step 8:
T8: Sample storage at -20’C
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3
4
5
Description of the action/
interactivity
Zoom-in tube with clear solution,
instruct user to store the tubes at 20’C into the freezer . Animate
opening the door of freezer, placing
the tube and closing the door. kindly
redraw the images.
Audio Narration
(if any)
The sample in rehydration buffer can be stored
at -20’C and can be thawed and used during
sample Quantification. Please go through the
future viewing IDD for more information.
Slide 58
Tab 01
Slide 911
Tab 02
Slide
12-14
Tab 03
Slide 19 Slide
20,21
Slide 1214
Tab 04
Tab 05
Tab 06
Name of the section/stage
Interactivity
area
Animation area
INTERACTION 1: In Slide-9: let user place the centrifuge tube in rotor
without balancing and proceeds with the setup.
Instruction: Display on monitor of a centrifuge error message saying
“Improper balancing” and let user stop the centrifuge, open the lid
and of the rotor to place the tube with same volume in each tubes
with balanced across the rotor and start the setup again.
Button 01
Button 02
Button 03
Instructions/ Working area
Credits
Slide
22,23
Tab 07
Slide
24-27
Tab 08
Tab 09
Tab 10
Tab 05
Tab 06
Tab 07
Name of the section/stage
Interactivity
area
Animation area
Button 01
Button 02
Button 03
Instructions/ Working area
Credits
APPENDIX 1
Questionnaire:
Question 1
What is the amino acid source in LB broth?
a) Glucose
b) Amino acid
c) Peptone
d) Nacl
Answer : Peptone
Question 2
What is the purpose of sonication?
a) To mix the culture
b) To denature the protein
c) To denature the
nucleotides
d) To lyse the cells
Answer: To lyse the cells
APPENDIX 1
Questionnaire:
Question 3
The reagent that separates the proteins and DNA-RNA from the cell is
a) Phenol
b) Glucose
c) Rehydration buffer
d) Trizol
Answer : Trizol
Question 4
What is the constituent(s) of the upper layer after trizol separation?
a) RNA
b) DNA and RNA
c) DNA,RNA and proteins
d) Proteins and DNA
Answer: RNA
APPENDIX 1
Questionnaire:
Question 5
The chemical that inactivates the RNases is
a) Trizol
b)Phenol
c)Guanidium –Hcl
d)Nacl
Answer : Guanidium -Hcl
APPENDIX 2
Links for further reading
Chen JH, Chang YW, Yao CW et al. Plasma proteome of severe acute respiratory
syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry.Proc
Natl Acad Sci U S A2004, 7;101(49):17039-44.
Eymann C, Dreisbach A, Albrecht D. A comprehensive proteome map of growing Bacillus
subtilis cells.
Proteomics. 2004 :2849-76.
Maldonado AM, Echevarría-Zomeño S, Jean-Baptiste S. et al. Evaluation of three different
protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by twodimensional electrophoresis. Proteomics 2008, 71(4):461-72.
2DE Tutorials by Angelika Görg : http://www.wzw.tum.de/blm/deg/
BOOKS
Biochemistry by Stryer et al., 5th edition
Biochemistry by A.L.Lehninger et al., 3rd edition
Biochemistry by Voet & Voet, 3rd edition
APPENDIX 3
Summary
The protein extracted should be devoid of the phenolics from the trizol reagent
which may hinder separation in IEF and SDS.
95%ethanol wash will remove such compounds from the sample
Sonication is more important step as it disrupts the cell which makes the proteins
to come out of the cell.
Care should be taken to prevent the unwanted particles in the extraction.