SDS-PAGE + Western Blot
Laboratory
What is Electrophoresis?
• Electrophoresis (to carry with electricity) is
the migration of charged molecules in an
electric field toward the electrode with the
opposite charge
• SDS-PAGE (Sodium dodecyl sulfate –
polyacrylamide gel electrophoresis) is a form
of electrophoresis that treats samples with
SDS to denature proteins
Why is SDS-PAGE used?
• How many proteins are in my sample?
• What are the molecular weights of the proteins?
• What differences are there in proteins from
different sources?
• How pure is my protein of interest?
Why are proteins separated on SDSPAGE?
• Small size of proteins
• Gel matrix of polyacrylamide is tighter than
agarose – resolve smaller molecules
• Polyacrylamide – small DNA
• Agarose – large protein
- Agarose gels will never resolve as tightly as
polyacrylamide gels
Two phases of polyacrylamide.
• Upper stacking gel = 4%
acrylamide
• Lower resolving gel = 15%
• Discontinuous system –
results in all proteins
separating or resolving at
same time
• Stacking gel – allow
proteins to migrate rapidly
and be compressed at edge
of denser resolving gel
• Separating gel – begin to
separate as per their
molecular weights
How are protein bands formed?
• Two ion fronts sandwich protein bands
• SDS-PAGE running buffer – Tris and glycine (pH
8.3)
• Gel – Tris-HCl buffer (pH 8.8)
• Chloride ions migrate rapidly than glycine ions
in an electric field
- When electrophoresis begins - proteins have
intermediate mobility - trapped between two
fronts -
How does polymerization start?
• Acrylamide and bis acrylamide monomers –
initiators – Ammonium persulfate (APS) and
catalyst – Tetramethylethylenediamine
(TEMED)
• Higher concentration of resolving gel is
poured – lower concentration stacking gel is
poured – sample comb is inserted into
unpolymerized stacking gel solution
Molecular weight of proteins
• Proteins – composed of 20 aa – 89 to 204
daltons
• Kilodalton (KD) – used for protein molecular
masses – 10 KD and 220 KD
Factors affecting mobility
• Electrical charge + mass = mobility
• SDS added in sample buffer and gel running
buffer
- Strong anionic (negatively charged) detergent
- Binds and coats proteins – denatured in linear
chains
- Mass determines migration
Denaturation of four structures of
proteins
• Primary, secondary,
tertiary and quaternary
• β-mercaptoethanol
(BME) or dithiothreitol
(DTT) – breaks disulfide
bonds
• Heat, reducing agent
and ionic detergent –
disrupt 2°, 3° and 4° results in linear chains
of aa
How to identify proteins in
polyacrylamide gel?
• Complex mixture of proteins
- Appear as distinct blue- stained bands(stained
gel)
- Positions and intensities – determine size and
abundance of proteins
- Specific proteins – Western blots – positive
identification by antibodies
Sources of errors
• Identical migration may be different proteins
of same sizes
• Proteins of similar composition, function and
evolutionary origin may be different in
molecular weight because of post
translational modifications
What are the different reagents that
will be used?
• Molecular weight markers – Precision plus
protein kaleidoscope prestained protein
standards
• Proteins genetically engineered to be specific
molecular weights then bound to dye
molecules – visible on gel
• Heated to 37 ° for 5 minutes
• Load 5 µl per gel
What are the different reagents that
will be used?
• Laemmli sample buffer – used to solubilize
proteins in fish muscle samples
- Mixture of Tris buffer + SDS + tracking dye
(bromophenol blue) + Glycerol
• Actin and Myosin standard – used to identify
conserved muscle proteins – positive control –
rabbit skeletal muscle consisting myofibrils
- Actin, myosin heavy chain, three myosin light
chains and tropomyosin- on destained gel
Actin and myosin standard
• Bands at 210 KD (myosin heavy) and 43 KD
(actin) – present in all of muscle samples
- Biologically active myosin has quaternary
structure - both heavy and light chains
- Myosin light chains (15-25 KD) vary in
molecular weight between fish species
What are the different reagents that
will be used?
• Dithiothreitol (DTT) or β-mercaptoethanol
(BME) – added to Laemmli buffer to ensure
complete breakage of disulfide bonds.
- Reduces formation of background bands
• Ready Gel precast gels – 15% Tris-HCl gels
- Resolves smaller molecular weight proteins
What are the different reagents that
will be used?
• Electrophoresis running (1X TGS) buffer –
Tris-glycine-SDS (TGS) running buffer contains
Tris to buffer the pH, glycine to provide ions to
transmit current, and SDS to maintain
denaturation of proteins in gel.
• Bio-Safe Coomassie protein stain – High
affinity between dye and proteins
- Wash gel 3 times with DI H2O before staining
What are the different reagents that
will be used?
• Bio-Safe Coomassie protein stain – Optimal
staining time is 1 hour – gentle shaking
- Destaining of gel – protein bands become
intense
- Rinse the stained gel with several changes of a
large volume of deionized water
- And complete destaining overnight in water
What are the different materials
needed for each workstation?
• Fish samples, Blade/knife, water bath set to
95 degree, Prot/Elec pipet tips for gel loading,
Mini-protean 3 electrophoresis module (gel
box), Power supply, gel comb, staining trays,
DI H2O, etc
Steps
• Protein extraction from various fish samples
(March 18) – 25 minutes- March 18
• Run SDS-PAGE (March 25) – 30 minutes
- Stain one gel with Coomassie blue (March 25
+ 26)
• Another gel – western blot (March 25)- 2.5 hrs
• Immunodetection with antibodies(March 27)
Western Blot reagents and equipment
• Mini Trans-Blot Apparatus : Passes electric
current horizontally through gel – forcing
negatively charged proteins to migrate out of gel
onto nitrocellulose membranes
• Nitrocellulose membranes: Proteins bind to
positive charge
- Durable membrane can undergo multiple
washing and incubation steps
- White background to visualize color development
of proteins
Western Blot reagents and equipment
• Blotting paper – supports gel and
nitrocellulose
- Protects filter pads during assembly and
electrophoresis
- Facilitates uniform flow of buffer and current
through gel
- 100% cotton fiber – does not contain any
additives that may interfere with blotting
process
Western Blot reagents and equipment
• Filter pads: Press the gel and nitrocellulose
tightly and uniformly
- Eliminate bubbles – allows transfer of proteins
out of gel onto the membrane
- Should be cleaned in distilled water before use
Western Blot reagents and equipment
• Blotting buffer: Should be diluted to 1X
• Blocker: 5% nonfat dried milk powder in
phosphate buffered saline (PBS) and 0.025%
tween 20
• Surface area unoccupied by proteins- blocked
by incubating milk solution – before
incubation with primary antibody
Western Blot reagents and equipment
• Primary antibody: recognizes or bind protein
under study
- Injecting chicken myosin protein into mice
• Secondary antibody: binds to primary
antibody
- Polyclonal goat anti-mouse antibody
conjugated to horseradish peroxidase enzyme
- Injecting goats with primary mouse antibodies
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This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the
U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity
employer and does not discriminate on the following basis:
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affiliation or belief; and
against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the
basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his
or her participation in any WIA Title I-financially assisted program or activity.
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