SDSPAGE

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IGP Methodology Course
SDS-PAGE
Sodium Dodecyl Sulfate- Polyacrylamide Gel
Electrophoresis
9.11.03
Jamie Hearnes Pietenpol Laboratory Biochemistry
Department jamie.hearnes_at_vanderbilt.edu
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Lecture Outline

• Background of Laemmli SDS-PAGE
• Function of SDS in this system
• Components of the discontinuous
• SDS-PAGE system
• Set-up and protocols for SDS-PAGE
• Protein staining
• Applications

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Native PAGE vs. SDS-PAGE
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Laemmli SDS-PAGE Method
The most popular method used today was developed
by U.K. Laemmli
Laemmli, U.K. 1970. Cleavage of structural
proteins during the assembly of the head of
bacteriophage T4. Nature 227680-685.
The Laemmli system is a modification of the
system that Orstein and Davis used to
fractionate serum proteins in a native
environment. L. Orstein. 1964. Ann. N.Y. Acad.
Sci. 121321. B. J. Davis. 1964. Ann. N.Y. Acad.
Sci. 121404.
Laemmli introduced SDS into the system.
Both Orstein and Davis system and Laemmlis
adaptation are examples of discontinuous gel
systems.
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Continuous vs Discontinuous Gel Systems
Discontinuous - A system that uses 2 continuous
but distinct gels that are made with different
buffers. In addition, the electrophoresis
buffer has a different conductivity than that of
the gel buffers.
Continuous - A system that has only a single
separating gel uses the same buffer in the tank
and the gel. Advantage versatile and
simple Disadvantage resolution is not as good
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How does the SDS work in this system?
SDS (sodium dodecyl sulfate) is an anionic
(negatively charged) detergent that binds to
proteins and disrupts non-covalent bonds. This
causes the protein to unfold and become
rod-like.
1.4 mg of SDS will be able to bind to every mg of
protein
The amount of SDS bound to the protein is
proportional to the MW of the protein and is
independent of the protein sequence, so the
proteins will migrate through the gel according
to size.
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How does the discontinuous SDS-PAGE system work?
Polyacrylamide
Polyacrylamide gels are formed by
copolymerization of acrylamide (CH2CH-CO-NH2)
and the crosslinking comonomer bisacrylamide
(CH2CH-CO-NH-CH2-NH-CO-CHCH2).
Polymerization is catalyzed by free radicals
generated from ammonium persulfate (APS), which
is the initiator.
Tetramethylethylenediamine (TEMED) is the
catalyst used to form free radicals from the APS.
Oxygen, a free radical scavenger, interferes with
polymerization.
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How does the discontinuous SDS-PAGE system work?
pore size
Polymerization of the bisacrylamide and
acrylamide creates a 3D network of fibers and
pores that acts as a sieve.
Increasing the amount of acrylamide in a gel will
decrease its pore size.
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How does the discontinuous SDS-PAGE system work?
2 types of gels used
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How does the discontinuous SDS-PAGE system work?
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General Characteristics of Polyacrylamide Gels
PAGE rigs are usually vertical, using the slab
gel format.
Thickness ranges from 0.5 mm to 3.0 mm
usually 0.75 - 1 mm is used The advantage of
thick gels is the increased protein
capacity. The disadvantage is that they are less
efficient at dissipating heat and they
are more difficult to stain and destain.
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Brands of Vertical Electrophoresis Equipment
BioRad
Novex
Hoefer
Owl Scientific
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Components of a Standard Vertical
Electrophoresis Apparatus
BioRad
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Assembly of a Standard Vertical Apparatus
Hoefer
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Assembly of a Standard Vertical Apparatus
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Gel Preparation
Most PAGE gels can resolve proteins that differ
in size by at least 3.
The stacker is usually 3 acrylamide
The separating gel can range from 5-15 depending
on the size of proteins needed to be resolved.
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Gel Preparation
CAUTION ACRYLAMIDE IS A CUMULATIVE
NEUROTOXIN! ALWAYS WEAR GLOVES AND BE VERY
CAREFUL!
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Gel Preparation

• Mix components of the separating gel (adding
  TEMED right
• before pouring)

• Pour liquid into the space between the glass
  plates and
• overlay with isopropanol.

• 30 min to 1 h after pouring the gel
  polymerization should be
• complete. The polymerization time is dependent
  upon
• acrylamide concentration and thickness of gel.

• Wash out the isopropanol with H2O and dry between
  the
• plates with filter paper or paper towels.

• Mix components of the stacker gel (again adding
  TEMED last)
• and pour into the space between the glass
  plates, filling it all
• the way to the top.

6. Insert comb, making sure there are no
bubbles in the well.
7. Allow to polymerize for at least 10 minutes.
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Pouring the Gels
Hoefer
BioRad
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Inserting the Comb
Hoefer
BioRad
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Additional Information
In addition to single percentage gels, gradient
gels can also be used. These types of gels
resolve high and low MW bands on the same gel.
Gradient gels are also used to determine the MW
of a protein.
Precast gels can be purchased commercially.
Advantages convenience Disadvantages
expense and expiration dates
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Sample Preparation

• After determining the concentration of protein in
  each sample,
• aliquot the amount of each sample needed into a
  new tube.

• Add concentrated sample loading buffer (SLB) to a
  final
• concentration of 1X SLB with all the samples
  having the same
• final volume.

• Heat the samples for 10 minutes at 85C. This
  helps to
• completely denature the proteins in the sample.

SDS -- breaks noncovalent bonds in proteins
?-mercaptoethanol -- reducing agent that breaks
disulfide bonds within and between proteins
Bromophenol blue dye -- allows the sample
migration to be visually monitored
Glycerol -- makes the sample sink into the wells
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Assembly of a Standard Vertical Apparatus
BioRad
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Assembly of the BioRad Rig
BioRad
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Loading of a Standard Vertical Gel
BioRad
Hoefer
Be careful to load each well slowly and carefully
to keep the samples from leaking into other
wells.
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Assembly of the Hoefer Rig
Hoefer
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Connecting the Rig to the Power Supply
BioRad
BioRad
Hoefer
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Components of the Mini Gel Apparatus
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Assembling a Mini Gel Apparatus
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Assembling a Mini Gel Apparatus
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Assembling a Mini Gel Apparatus
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Assembling a Mini Gel Apparatus
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Proteins Used as Molecular Weight Standards
Spector, D., Goldman, R., and Leinwand, A. 1998.
Cells A Laboratory Manual Vol 1. New York
Cold Spring Harbor Laboratory Press.
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Rainbow
Regular
200 kD
250 kD
Protein Molecular Weight Markers
10 kD
10 kD
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Running an SDS-polyacrylamide Gel
Most commercial equipment is color coded.
RED () terminal -- goes to lower buffer
chamber BLACK (-) terminal -- goes to
upper buffer chamber
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Running an SDS-polyacrylamide Gel
Most SDS-PAGE separations are performed under
constant current (mA).
Ohms law current x resistance voltage
I x R V
The resistance of the gel increases during the
run. If the current is constant, then the
voltage will increase as the resistance increases.
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Running an SDS-polyacrylamide Gel
Power Supplies These usually have more than
one pair of outlets. The pairs (red and black)
are connected in parallel with each other
internally.
In a parallel circuit, the voltage is the same
across each gel, but the total current is the sum
of the individual currents.
If using constant current, the setting has to be
increased for each gel connected to the power
supply.
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Running an SDS-polyacrylamide Gel
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Separating Proteins of Small Molecular Weight
The Laemmli method uses the Tris-glycine buffer
system and can only resolve proteins down to 10
kD. The reason for this is due to the SDS
migrating with the small proteins. To resolve
proteins/peptides that are smaller than 10 kD the
Tricine system is usually used.
The Tricine system was developed by Schagger and
von Jagow in 1987, and it uses tricine as the
trailing ions instead of glycine. In this
environment the small proteins stack separately
from the SDS and can be resolved.
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Staining Proteins in SDS-PAGE Gels
Coomassie Brilliant Blue -- most common, least
sensitive --This is a triphenylmethane textile
dye that binds nonspecifically to proteins.
--Detection limit 100ng of protein
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Staining Proteins in SDS-PAGE Gels
Copper/Zinc -- rapid, reversible --These bind
to components in the polyacrylamide gels but
not to the SDS, so the proteins do not
stain. --The result is a negatively stained gel
where the proteins are clear bands.
Protein is visualized against a black
background. --With this method proteins are not
permanently fixed --Detection limit
intermediate
copper
zinc
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Staining Proteins in SDS-PAGE Gels
Silver -- most sensitive --Process relies on
differential reduction of silver ions that
are bound to the side chains of proteins.
--Can use either ammoniacal silver or silver
nitrate. Silver nitrate is easier to
prepare and doesnt generate potentially
explosive products. --Commonly used to assay
protein purity --Detection limit 0.1-1.0 ng
of protein --CAUTION Be sure to wear gloves
and handle the gel gently because pressure
and fingerprints will yield staining
artifacts.
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Staining Proteins in SDS-PAGE Gels
Fluorescent -- --Different colored fluorescent
reagents can be used. They are visualized
by UV light. --These reagents bind to SDS (not
the protein) so they dont mask antigen
recognition sites. --Detection limit rivals
silver stain
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Possible Applications of SDS-PAGE
Establishing molecular weights of
proteins Determining sample purity Identifying
disulfide bonds Quantifying proteins Blotting
applications Protein identification Etc.