Methodology for the extraction of Bacterial protein

1
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
• gt Prior Viewing IDD2 Plant extraction, IDD6
  Serum extraction
• gt Future Viewing IDD11 Protein
  Quantification, IDD 12 Rehydration, IDD 15 IEF,
  IDD17 SDS-PAGE, IDD20 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-ShareAl
ike 2.5 India license
2
Learning objectives
1

• After interacting with this learning object, the
  learner will be able to
• Identify the mechanism in TRIzol extraction
• Examine the technique involved in culture growth
• Interpret the results of the experiment
• Assess the troubleshooting steps involved in the
  experiments

2
3
4
5
3
Master Layout
1
Inoculation of Bacterial culture (Slide 5-8)
Centrifugation followed by Buffer treatment
(Slide 9-11)
2
Cell Lysis by sonication (Slide 12-14)
Trizol treatment (Slide 12-14)
3
Chloroform treatment (Slide 19)

• Absolute alcohol treatment (Slide 20-21)

• Acetone precipitation (Slide 22-23)

4
Sample treatment with rehydration buffer
(Slide 24-27)
Store the sample at -20C (Slide 28)
5
4
Definitions and Keywords
1
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 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-Dimethyl3-(4-5,9,16-tri
hydroxy-2,15-dimethyltetracyclo8.7.0.02,7.011,15
heptadecan-14-ylpentanamido)propylazaniumylpro
pane-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.
2
3
4
5
5
Step 1
T1Inoculation of Bacterial culture
1
2
Video File LAMINAR air flow
3
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.
4
5
6
Step 1
T1Inoculation of Bacterial culture
1
LB Broth
2
3
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.
4
5
7
Step 1
T1Inoculation of Bacterial culture
1
2
3
Place the inoculated tube in the shaker-incubator
at 37C for 6-8 hours.
4
5
8
Step 1
T1Inoculation of Bacterial culture
1
2
3
Transfer the bacterial culture into the clean
centrifuge tube under aseptic condition for
further processing.
4
5
9
Step 2
T2 Centrifugation followed by Buffer treatment
1
2
3
Centrifuge the culture for 10 min at 12000 rpm
maintaining at 4C to harvest/collect the
culture.

• .
• .

4
5
10
Step 2
T2 Centrifugation followed by Buffer treatment
1
2
3
Remove the supernatant completely without
disturbing the pellet, now take the pellet for
further processing.

• .

4
5
Video File Centrifuge.MTS and Centrifuge_part2.MT
Sc
11
Step 2
T2 Centrifugation followed by 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.

• .

4
5
12
Step 3
T3 Cell Lysis by 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 helps
protein extraction by cell lysis.
4
5
Video File Sonication
13
Step 3
T3 Cell Lysis by 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. Please redraw the figure
4
5
14
Step 3
T3 Cell Lysis by sonication
1
2
3
Centrifuge the contents to remove the debris and
collect the supernatant for further processing.

• .

4
5
15
Step 4
T4 Trizol treatment
1
2
3
Trizol reagent consists of guanidium thiocyanate,
phenol,chloroform that separates DNA, RNA and
proteins from each other within the solution.
4
5
16
Step 4
T4 Trizol treatment
1
2
3
Add trizol to the supernatant and vortex it
thoroughly.
4
5
Video File Vortex
17
Step 4
T4 Trizol treatment
1
Chloroform
2
3
Add chloroform to the sample, mix thoroughly till
the color appear like milk-shake and keep at room
temperature for 5min till you get the phase
separation.
4
5
18
Step 4
T4 Trizol treatment
1
2
3
Three layers are formed after centrifugation. The
top layer containing RNA, Middle layer with
protein and the bottom layer with DNA.
4
5
19
Step 5
T5 Absolute alcohol treatment
1
2
3
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.
4
5
20
Step 5
T6 Acetone treatment
1
2
3
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.
4
5
21
Step 5
T6 Acetone treatment
1
2
3
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.
4
5
22
Step 6
1
T7wash buffer preparation
2
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
0.000
During the material measurement on paper, the
weight of the paper need to be tarred or set to
zero before weighing.
4
Video File Measuring Balance
5
23
Step 6
1
T7wash buffer preparation
Guanidine-HCL
2
ethanol
Audio Narration
Description of the action
3
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.
Prepare wash solution, washing is carried to
remove excess broth and other contaminants if
present in the solution.
4
5
24
Step 6
1
T7 buffer preparation
CHAPS
2
Rehydration buffer (RB)
water
urea
3
Audio Narration
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 slide22, 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.
Weigh 0.6g of urea, 0.02g of CHAPS to prepare
rehydration buffer which must be store at 4C.
4
5
25
Step 7
T8 Pellet washing
1
2
3
Remove the supernatant carefully and air dry the
pellet. Pellet retains the pink color, carry out
washing the pellet with 0.3 M guanidium HCl in
95 ethanol for 4 times to remove the color and
for inactivation of RNAses. Each pellet wash need
to be followed by centrifugation by discarding
the supernatant till pellet becomes color-less.
4
5
26
Step 8
T8 Rehydration buffer treatment
1
2
3
The rehydration buffer consists of CHAPS
used to solubilize the proteins including
membrane proteins. Urea used to denature protein
structure.
4
5
27
Step 7
T8 Rehydration buffer treatment
1
2
3
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.
4
5
28
Step 8
T8 Sample storage at -20C
1
2
3
The sample in rehydration buffer can be stored at
-20C and can be thawed and used during sample
Quantification. Please go through the future
viewing IDD for more information.
4
5
29
Slide 19
Slide 9-11
Slide 12-14
Slide 12-14
Slide 20,21
Slide 5-8
Tab 01
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Name of the section/stage

• 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.

Interactivity area
Instructions/ Working area
Credits
30
Slide 24-27
Slide 22,23
Tab 08
Tab 09
Tab 10
Tab 05
Tab 06
Tab 07
Tab 07
Name of the section/stage

• Animation area

Interactivity area
Instructions/ Working area
Credits
31
Questionnaire
APPENDIX 1
32
Questionnaire
APPENDIX 1
33
Questionnaire
APPENDIX 1
34
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
35
APPENDIX 3
Summary
The protein extracted should be devoid of the
phenolics from the trizol reagent which may
hinder separation in IEF and SDS. 95ethanol
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.