Related LOs: Sonication

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Effect of Sonication on bacterial and Serum
protein extraction
Sonication is the process by which the high
energy sound waves causing cell lyses, rupture of
cell wall, causes release of contents from the
cell. The method is more effective in cell
disruption. Due to its high efficiency the method
is highly suitable fro the protein extraction
from the cell and the serum samples

• Related LOs Sonication
• gt Prior Viewing IDD-1. Extraction of
  bacterial protein, IDD-6. Extraction of serum
  protein.
• gt Future Viewing IDD-11. Protein
  quantification, IDD-14. Isoelectric focusing,
  IDD-17. SDS-PAGE
• Course Name Sonication on bacteria and serum
  protein extract
• Level(UG/PG) UG
• Author(s) Dinesh Raghu, Vinayak Pachapur
• Mentor Dr. Sanjeeva Srivastava

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Learning objectives
1

• The study helps the listener to learn
• Define the mechanism of cell lysing by sonication
• Operate the steps involved in the extraction
  technique
• Outline the precautions to be followed during the
  sonication.
• Infer the steps involved to perform experiment.
• Assess the troubleshooting steps involved in the
  experiments.

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Master Layout
1
Serum Sample processing (Slide4)
2
Buffer Treatment (Slide 5-6)
Sonication (Slide 7-10)
3
Bacteria Sample processing (Slide11-12 )
4
Buffer Treatment (Slide 13)
5
Sonication (Slide 14-17)
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Step 1
T1Sample processing
1
2
3
Remove the serum from -80 C and allow it to thaw
by keeping it on ice for 15 min. Transfer the
required amount of the serum to the fresh, clean
eppendorf tube for further sample processing.
Transfer of sample must be a quick process.
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5
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Step 2
1

• T2Buffer addition

2
3
Dilute the serum 5 times using phosphate buffer
of pH 7.4, which helps to maintain the buffer
during the sonication step.
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5
6
Step 3
1

• T2Buffer addition

2
3
Vortex the sample to achieve uniform mixing
Vortex the sample to achieve uniform mixing.
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Step 3
1
T3Sonication
2
3
Keep the sample on ice and start sonication by
providing 6 cycles of pulses for 5sec, 20
amplitude with 5sec gap. Sonication carries out
cell lysis which helps for protein extraction.
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5
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Step 3
T3Sonication
1
sonication
2
3
High frequency sound waves split the aggregated
serum proteins and thereby makes the sample easy
for further analysis
Zoom in a aggregation as in the figure from the
tube. Show the sound waves hitting the those
aggregation and causing separation/ splitting
of aggreagated protein and the release of
contents. Please redraw the figure
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5
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Step 3
1
T3Sonication
2
3
After sonication, the cells are lysed now serum
contains high abundance proteins like, albumin,
IgA, IgG, haptoglobin along with low abundance
protein. High abundance protein will interfere in
gel separation and prevent the separation of low
abundance proteins. After sonication steps the
sample is taken for further purification process.
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10
Step 3

1
Purification steps same as IDD-6. Extraction of
serum protein from Slide 17-32. For more
information on the extraction steps go through
future viewing IDD.
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3

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Step 1
T1 Sample preparation (bacterial)
1
2
3
Transfer the bacterial culture into the clean
centrifuge tube under aseptic condition.
Centrifuge the culture for 10 min at 12000 rpm
maintaining at 4C
4
5
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Step 2
T1 Sample preparation (bacterial)
1
2
3
Remove the supernatant completely without
disturbing the pellet, now take the pellet for
further processing.
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5
13
Step 2
T2 Buffer treatment
1
2
3
Wash the pellet with phosphate buffer thoroughly
to remove the excess broth. Once broth is removed
completely, cell lysis need to be carried out.
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5
14
Step 3
T3 Sonication
1
2
3
Keep the sample on ice and start sonication by
providing 6 cycles of pulses for 5 sec ,20
amplitude with 5 sec gap. Sonication carries out
cell lysis which helps for protein extraction.
4
5
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Step 3
T3 Sonication
1
2
3
High frequency sound waves break open the cell
wall and the contents are released into the buffer
Zoom in a cell from the tube. Show the sound
waves hitting the cell and causing cell lyses and
the release of contents. Animate cell breaking
with release of content into the tube from inside
the cell. Please redraw the figure
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5
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Step 3
T3 Sonication
1
2
3
Centrifuge the contents to remove the debris and
collect the supernatant for further processing.
4
5
17
Step 3
1
Extraction steps are same as IDD-1. Extraction of
bacterial protein from Slide 16-32 For more
information on the purification steps go through
future viewing IDD.
2
3

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5
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Slide 11-12
Slide 14-17
Slide 13
Slide 4
Slide 7-10
Slide 5-6
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage BACTERIAL CULTURE
Animation area Interaction 1 slide-14 user
carrying out sonication without cooling on ice
bath, no gap between the bursts and proceeds
further. Instructions let user place the tube
on ice packs at the time of sonication and gives
a time delay for few seconds between the
bursts. Interaction 2 slide-14 user carrying
out sonication for longer time. Instructions
animate the tube getting hot and starts melting.
Excessive sonication will lead to heating and ice
packs helps down in cooling the sample.
Interactivity area
Instructions/ Working area
Credits
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Questionnaire
APPENDIX 1

• Question 1
• Amplitude used for the sonication of serum is
• 20
• 30
• 40
• 50
• Answer 20

What is the purpose of sonication in bacterial
protein extraction?

• To mix the culture
• To denature the protein
• To denature the nucleotides
• To lyse the cells
• Answer To lyse the cells

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Questionnaire
APPENDIX 1
What is the purpose of sonication in serum
protein extraction?

• To mix the culture
• To deplete high abundance protein
• To denature the nucleotides
• To break the protein aggregates
• Answer To break the protein aggregates

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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, 7101(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
two-dimensional 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
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APPENDIX 3
Summary
Ultrasonic waves generated by a sonicator lyse
cells through shear forces. Complete shearing
is obtained when maximal agitation is achieved,
but care must be taken to minimize heating and
foaming. Sonicate cell suspension in short
bursts to avoid heating.Cool on ice between
bursts. Sonication is a vigorous way of cell
lysis.