Transcript SDS_PAGE Intro - Advances in Bioscience Education (ABE)

ABE Workshop 2006
The SDS-PAGE
Dongping Lu
Dongping Lu
ABE Workshop
June 21, 2006
Western blotting
Trouble shooting
Absorption value
1.
We should have
a parameter: Total
protein /plant tissue
2.
Use higher
concentration of BSA
Standard, like BSA
10mg/ml
BSA
Today’s work
Run the SDS-PAGE gel:
 Transfer the protein from gel to nitrocellulose
membrane
 Stain the gel with Coomassie Blue
group 1 & 4: wt and pdi2 mutant
group 2 & 3: wt and gfp-2sc
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PAGE
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Gels are cast by polymerizing a solution of acrylamide
monomers into polyacrylamide chains
Gel pore size can be varied by adjusting the concentrations
of polyacrylamide
Smaller proteins migrate faster than larger proteins through
the gel
Native proteins
SDS (sodium dodecyl sulfate) binds to and
coat the protein
SDS
1. SDS disrupts some of the noncovalent
interactions that stabilize protein
quaternary and tertiary structures,
facilitates denaturation.
2. SDS also has a negative electrical
charge and binds to proteins in a constant
mass ratio of 1.4 : 1, so that the total
amount of detergent bound is directly
proportional to the molecular weight of
the protein.
3. The ‘coating’ of negatively charged
SDS overwhelms the inherent charges of
protein molecules and gives them a
uniform charge to mass ratio.
4. This allows proteins to be separated on
the basis of their relative sizes,
SDS
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all polypeptide chains are then forced into extended
conformations
SDS treatment eliminates the effect of differences in
shape
individual polypeptide chains migrate as a negatively
charged SDS-protein complex through the porous
polyacrylamide gel
speed of migration is proportional to the size of the proteins

smaller polypeptides running faster than larger polypeptides
How about covalent link?
DTT/Me
SH
S-S
HS
Noncovalent
covalent
Heating the sample
Heating your samples at 99ºC completed denaturation of
the protein molecules, ensuring that they were in completely
linear form.
 This allowed SDS to bind all regions of each protein
equally.
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Protein loading buffer
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Protein gel loading buffer contains Tris buffer to
maintain constant pH
glycerol to increase sample density,
the strong ionic detergent SDS (sodium
dodecylsulfate),
β-mercaptoethanol, a reducing agent. . Betamercaptoethanol eliminates disulfide bonds in
proteins by reducing them (adding hydrogen
atoms).
Heating
Running the gel
Stacking gel
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To obtain optimal resolution of proteins, a “stacking” gel is
poured over the top of the “resolving” gel.
The stacking gel
lower concentration of acrylamide (larger pore size),
lower pH
different ionic content
This allows the proteins in a lane to be concentrated into a
tight band before entering the running or resolving gel
produces a gel with tighter or better separated protein
bands
Transfer the protein from the gel to the
membrane
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Transfer of the proteins fractionated by SDSPAGE to a solid support membrane (Western
blotting) can be accomplished by electroblotting
Transfer
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In this procedure, a sandwich of
gel and solid support membrane
(Nitrocellulose or PVDF) is
compressed in a cassette and
immersed in buffer between two
parallel electrodes.
A current is passed at right angles
to the gel, which causes the
separated
proteins
to
electrophorese out of the gel and
onto the solid support membrane
Gel staining
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Once proteins have been fractionated by
electrophoresis, to make them visible, staining with
a material that will bind to proteins but not
polyacrylamide.
the most common one: staining with Coomassie
Blue.
This is a dye that binds most proteins uniformly
based on interactions with the carbon-nitrogen
backbone.
The dye is dissolved in a solution that contains both
methanol and acetic acid
gel-drying frames
for drying of SDS-PAGE gels
Gel drying
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SDS-PAGE gels between two moistened
sheets of Gel Drying Film (from Promega)
on the bench.
Clamp the Gel Drying Frame
Dry over night
It is important to remove all the air bubbles
from between the two sheets of gel drying
films. Air bubbles may cause the gel to
crack during drying