Transcript Online Counseling Resource YCMOU ELearning Drive…

Online Counseling Resource
YCMOU ELearning Drive…
School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India
SEP-SBI084-CP01-03
Introduction
Programmes and Courses
 SEP-SBI084-CP01-U01
School of Science and Technology, Online Counseling Resource…
Credits
 Academic Inputs by
 Sonali Alkari
 Counsellor, YCMOU Nagpur Study Centre,
 Faculty LAD college P.G. D of Biotechnology

Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]
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School of Science and Technology, Online Counseling Resource…
How to Use This Resource

Counselor at each study center should use this presentation to deliver
lecture of 40-60 minutes during Face-To-Face counseling.

Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.

Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.

Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.

Appear several times, for all the Self-Tests, available for this course.

Student can use handouts for last minutes preparation just before end
exam.
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School of Science and Technology, Online Counseling Resource…
Learning Objectives
After studying this module, you should be able to :
 Explain what is dialysis.
 State principle and technique of dialysis.
 State Factors affecting electrophoresis
 State nature of dialysis membrane
 Explain principle and technique of salting in
 Explain principle and technique ofsalting out
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Dialysis:1
 Dialysis provides a means to change the buffer
solution for a protein sample.
 In dialysis, a protein solution is placed inside a bag
consisting of a semi-permeable membrane.
 The sample is then dialyzed against several
changes of the desired final buffer.
 First, the concentrated protein solution is placed in
dialysis bag with small holes which allow water and
salt to pass out of the bag while protein is retained.
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Dialysis:2
 Next the dialysis bag is placed in a large
volume of buffer and stirred for many hours
(16 to 24 hours), which allows the solution
inside the bag to equilibrate with the
solution outside the bag with respect to salt
concentration.
 When this process of equilibration is
repeated several times (replacing the
external solution with low salt solution each
time), the protein solution in the bag will
reach a low salt concentration:
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Dialysis:3
This graphic illustrates the dialysis process.
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Dialysis:4
The graphic illustrates the complete dialysis
process, except for it suggests you do this
with distilled water.
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Dialysis:5
 Buffer is used in this process to prevent the protein
from denaturing due to the fact that distilled or
deionized water is too low in salt
 and may have an undesirable pH for your protein,
which may cause it to denature.
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Dialysis of Purified Protein:1
1. Wear gloves any time you are doing a dialysis.
2. Get a large 2000ml beaker or larger if necessary
3. Estimate the volume of your sample and make a
100 fold excess amount of 10X buffer (If your
sample volume is 10ml then make 1000ml of 10X
buffer)
4. Place the dialysis buffer in the large beaker, cover
with saran wrap, and place in the cold room until
further use.
5. Get 2 dialysis clamps
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Dialysis of Purified Protein:2
6. Get enough dialysis bag to hold all of your sample.
Each millimeter can hold about 1ml so measure out
a length equal to the volume of your sample.
7. Get a new clean razor blade and make one clean
cut at the end of the length you re going to use.
8. Rinse the orange clamps and the dialysis bag
excessively with dH2O. Rinse the dialysis bag very
well so that it becomes hydrated.
9. Once you have hydrated the dialysis bag you can
roll your fingers along one of the ends until it
opens.
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Dialysis of Purified Protein:3
10.Once you have one end of the dialysis bag open
run lots of dH2O through it to rinse the inside and
fully hydrate it.
11.Take one of the orange clamps and clamp one of
the ends so that it is closed shut.
12.Fill the entire dialysis bag with water so that you
can look for leaks. Apply a little pressure to the
bag to look for small holes and to make sure that
the clamp is working properly.
13.Grab the test tube samples from the cold room that
you have decided to use according to your gel.
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Dialysis of Purified Protein:4
14. Use a powered pipette to collect all the fractions
that you plan to dialyze and put the entire sample
into the dialysis bag.
15. Take one of the orange clamps and clamp one of
the ends so that it is closed shut.
16. Fill the entire dialysis bag with water so that you
can look for leaks. Apply a little pressure to the
bag to look for small holes and to make sure that
the clamp is working properly.
17. Grab the test tube samples from the cold room
that you have decided to use according to your
gel.
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Dialysis of Purified Protein:5
18. Clamp the other side of the dialysis bad with the
other orange clamp leaving a little room at the end
so that the bag is easy to grab and work with.
19. Dialyze for three changes of buffer each going over
8 hours.
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Dialysis Membrane:1
 The dialysis membrane is made of cellulose acetate
and looks and feels like Saran wrap.
 It is semi-permeable, meaning that molecules
below a specified molecular weight can readily
pass through the membrane whereas larger
molecules cannot.
 Dialysis membranes are available with a wide
variety of molecular weight cutoffs; 10,000 and
40,000 dalton membranes are typically used for
protein dialysis.
 Once you have one end of the dialysis bag open
run lots of dH2O through it to rinse the inside and
fully hydrate it.
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School of Science and Technology, Online Counseling Resource…
Dialysis Membrane:2
 Sometimes, proteins will precipitate during
the dialysis process and may need to
centrifuge the solution after dialysis to
remove any particles which would interfere
with the next step - such as ion-exchange
chromatography where particles would clog
the
column
and
prevent
the
chromatography step from working.
 In addition, you may lose enzyme activity
during dialysis.
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Salting Out:1
 This is the most common way to precipitate
proteins.
 Protein solubility is a complex function of the
physiochemical nature of the proteins, pH
temperature and the concentration of the salt used.
 The process of salting out, the separation of an
organic phase from an aqueous phase by the
addition of a salt has been known for nearly a
century.
 This method is commonly used by biochemists in
the purification of proteins.
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School of Science and Technology, Online Counseling Resource…
Salting Out:2
 The most common type of precipitation for proteins
is salt induced precipitation.
 Protein solubility depends on several factors.
 It is observed that at low concentration of the salt,
solubility of the proteins usually increases slightly.
 This is termed Salting in.
 But at high concentrations of salt, the solubility of
the proteins drops sharply.
 This is termed Salting out and the proteins
precipitate out.
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Salting Out:3
 It also depends on whether the salt is
Kosomtropic (stabilizes water structure) or
Chaotropic (disrupts water structure).
 At low concentrations of salt, solubility of
the proteins usually increases slightly
(salting in).
 But at high concentrations of salt, the
solubility of the proteins drop sharply
(salting out).
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Salting Out:4
 Initial salting in at low concentrations is explained
by the Debye-Huckel theory. Proteins are
surrounded by the salt counter ions (ions of
opposite net charge) and this screening results in
decreasing electrostatic free energy of the protein
and increasing activity of the solvent, which in turn,
leads to increasing solubility.
 This theory predicts the logarithm of solubility to be
proportional to the square root of the ionic
strength.
 The behavior of proteins in solutions at high salt
concentrations was explained by Kirkwood.
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Salting Out:5
 The abundance of the salt ions decreases the
solvating power of the salt ions decreases the
solubility
of
the
proteins
decreases
and
precipitation results.
 At high salt concentrations, the solubility is given
by the following empirical expression due to Cohn.
 log S = B - KI
 where S is the solubility of the protein, B is a
constant (function of protein, pH and temperature)
K is the salting out constant (function of pH, mixing
and salt), and I is the ionic strength of the salt
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Salting Out:6
 The log of protein solubility versus the salt
concentration.
 Different lines represent different proteins. Hence, by
using the appropriate concentration range of the
given salt, we can precipitate the protein of interest,
preferentially form a mixture of proteins.
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Salting Out:7
 The slope of the salting out curve is a function of the
protein and salt involved, but it is not a function of
the pH and temperature.
 Also, as the molecular weight of the protein
increases, the amount of salt required for
precipitation decreases.
 There is a series of the relative effectiveness of
different anions in the salts used for protein
precipitation.
 This is referred to as the lyotropic of Hofmeister
series.
 The order is citrate > phosphate > sulphate >
acetate which is about as good as chloride > nitrate
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> thiocyanate. © 2007, YCMOU. All Rights Reserved.
School of Science and Technology, Online Counseling Resource…
What You Learn…
You have learnt :
 Dialysis provides a means to change the buffer solution for
a protein sample.
 Buffer is used in this process to prevent the protein from
denaturing.
 The dialysis membrane is made of cellulose acetate and it
is semi-permeable in nature.
 Salting out is the most common way to precipitate
proteins.
 At low concentrations of salt, solubility of the proteins
usually increases slightly (salting in).
 But at high concentrations of salt, the solubility of the
proteins drop sharply (salting out).
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School of Science and Technology, Online Counseling Resource…
Critical Thinking Questions
1. State why buffer is used in protein dialysis.
2. Explain the procedure of protein dialysis in
details.
3. Give a detail account on salting in and
salting out.
4. What is the role of dialysis membrane?
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© 2008,
2007, YCMOU. All Rights Reserved.
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Hints For Critical Thinking Question
1. To prevent the protein from denaturing.
2. After placing , a protein solution is placed inside a
bag consisting of a semi-permeable membrane,
how buffer is changed.
3. solubility of the
concentration.
4. Selective passage
membrane.
proteins
of
at
molecules
© 2007, YCMOU. All Rights Reserved.
low
and
through
high
the
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Study Tips:1
 Book1
 Title: Biophysical
techniques )
Chemistry
(principles
and
 Author: Upadhay. Upadhay.Nath
 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
to
 Author: Freifelder
 Publisher: W. H. Freeman and Company
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School of Science and Technology, Online Counseling Resource…
Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics

Author: Roy R.N.
 Publisher:New Central Book Agency
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School of Science and Technology, Online Counseling Resource…
Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia
http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia
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End of the Presentation
Thank You !