Protein electrophoresis:
Introduction to SDS-PAGE
Aim:
-Separation of proteins in an electric field by electrophoresis.
Purposes:
-Estimation of molecular masses
-Relative abundances of major proteins in a sample
SDS-PAGE = Sodium Dodecyl Sulfate PolyAcrylamide Gel
Electrophoresis.
Uses a polyacrylamide gel as a support medium and SDS to
denature proteins.
Also called the Laemmli method.
Laemmli UK, Nature 1970, 227 (5259):680-5. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4..
Laemmli UK, Nature 1970, 227
(5259):680-5. Cleavage of structural
proteins during the assembly of the
head of bacteriophage T4..
http://www.bio-rad.com/LifeScience/pdf/Bulletin_1156.pdf
SDS is an anionic detergent.
A polypeptide chain binds SDS in molar proportion to its mass.
SDS denatures most complex structures of proteins. It is also
attracted toward the positively-charged anode.
About 1 SDS binds per 2 AA.
Polyacrylamide gels restrain larger molecules from migrating as fast
as smaller ones.
The separation of proteins depends almost entirely on the
differences between the molecular masses of polypeptides.
In a uniform gel, the migration distance of a protein (Rf) is
proportional to the -log of its mass.
Proteins of known masses are run along with the unknowns. This
allows to plot “Rf vs. mass”, and to estimate the masses of unknown
proteins.
Stacking gel
Separating (or
Resolving) gel
-
+
Lighter protein
Heavier protein
Polyacrylamide gels for SDS-PAGE
Important parameters:
-relative percentages of acrylamide and crosslinker (e.g. bis)
-gel dimensions
“Manual” preparation requires casting two different layers of
acrylamide between glass plates.
Lower layer: separating gel.
Upper layer: stacking gel with sample wells. Stacks proteins into
micrometer thin layers.
SDS Gels
-A 7% bis in acrylamide gel separates proteins with masses between
45 and 200 kDa (no resolution below 45 kDa).
-A denser gel, e.g. 14%, resolves smaller polypeptides. It would not
separate proteins above 60 kDa.
-To analyze the entire profile of a sample containing heavy and light
polypeptides: run two gels, or a density gradient gel.
ww2.chemistry.gatech.edu
Running Gel:
Buffer stock solution: 0.4% SDS, 0.5 M Tris-HCl @ pH 6.8.
Buffer, linker and acrylamide stock solutions are mixed to obtain from 7
to 15% of bisacrylamide.
Acrylamide polymerizes in the absence of O2: the mixture is placed
under vacuum for 5 min.
Then polymerization occurs quickly. The solution is poured into a
cassette.
The gel mix is poured, then overlaid with water-saturated butanol.
This will produce a smooth, leveled surface to keep bands straight
and uniform.
Stacking gel
•Stackers have 3-4.5% acrylamide (low density) to enable very large
proteins to run.
•At polymerization, the mix is poured into the cassettes on top of the
separating gel.
•A comb is inserted and adjusted evenly.
http://www.manufacturingchemist.com/news/article_page/title/46647
proteabio.com
Protein + sample denaturation
Protein denaturation prior to SDS-PAGE
Sample is mixed with 2% SDS, 20% glycerol, 20 mM Tris-Cl, pH 8, 2
mM EDTA, dithiothreitol (DTT), and bromophenol blue as a tracking
dye.
Roles of denaturing components
EDTA chelates divalent cations, then reduces the activity of enzymes
that require Ca2+ and Mg2+ ions.
Glycerol makes the sample denser than the buffer: sample will
remain at the bottom of a well rather than float out.
Dye: tracks the progress of electrophoresis.
SDS: adds negative charge to the amino acids. Proteins are
“straightened”, thus functionless.
DTT breaks covalent disulfide bonds.
Cystine contains a sulfhydryl (-SH) group that spontaneously forms
a disulfide bond (-S-S-) with another sulfhydryl group under normal
intracellular conditions.
DTT is a strong reducing agent for disulfide bonds.
Amounts to loadA typical mini-gel well holds 10 µL of a 2 mg/mL solution of
denatured protein (0.02 mg or 20 µg).
SDS-PAGE yields reproducible results, but provides low
accuracy for MW determination.
Running gels
The anode (+) must be connected to the bottom chamber and the
cathode (-) to the top chamber.
Gels are run at a voltage (typically 150 V) that will bring the
tracking dye to the bottom as quickly as possible without
overheating the gels.
Disassembly and staining
When the dye front is near the
bottom of the gel, the run is
stopped.
Plates are separated and the
gel is dropped into a staining
dish.
Staining usually requires
incubation overnight, with
agitation.
Staining protein gels
Commonly used: 0.1% Coomassie Blue dye in 50% methanol, 10%
glacial acetic acid.
Acidified methanol precipitates the proteins.
The dye inflitrates the entire gel, however only sticks permanently to
proteins.
Excess dye is washed out by 'destaining' with acetic acid/methanol,
also with agitation.
Gels can be dried down, cut or photographed for later analysis and
documentation.
Steps to analyze the gel
Calibrate the gel using standards of known molecular mass
Estimate molecular mass for each band of interest
Note differences in intensity of staining that reflect abundance of
individual proteins
Note unusual patterns that might indicate incomplete denaturation,
degradation, etc.
Estimate apparent molecular mass for unknowns
The relationship between mass and Rf is logarithmic. Data are
interpolated from the standard curve.
High molecular mass:
-myosin (205,000) - from rabbit muscle
-beta-galactosidase (116,000) - from Escherichia coli
-phosphorylase B (97,400) - from rabbit muscle
-bovine serum albumin (66,000)
-egg albumin (45,000)
-carbonic anhydrase (29,000) - from bovine erythrocytes
Low molecular mass
-bovine serum albumin 66,000
-egg albumin 45,000
-glyceraldehyde-3-phosphate dehydrogenase (36,000) - from
rabbit muscle
-carbonic anhydrase (29,000)
-trypsinogen (24,000) - from bovine pancreas
-soybean trypsin inhibitor (20,100)
-alpha-lactalbumin (14,200) - from bovine milk
205k
116k
97.4k
66k
45k
36k
29k
24k
20.1k
Measuring relative mobility of protein bands
Relative mobility is the distance migrated by a band divided by the
distance migrated by the dye front.
It is used to compare the migration of a protein from gel to gel,
regardless of the physical length of the gel or duration of
electrophoresis.
How gel concentration affects relative mobility
Acrylamide concentration determines the cut-off at the low end.
All polypeptides below a minimum mass run with the tracking dye.
A gel with low density resolves larger polypeptides while cutting off
lighter ones.
A gel of higher density will reveal smaller polypeptides, while
compressing and possibly distorting the larger ones.
3 = phosphorylase B (92,000)
Summary
SDS-PAGE: advantages
Relatively inexpensive
Low voltage used: easy to handle
Visual result
Makes comparison of samples easy
Possible to cut protein band from gel for further use
Disadvantages:
Mass determination not accurate
Results subject to experience of experimentalist
Stain may be difficult to remove for further use
Gel storage in liquid
Quantitation not accurate
1. a) The following standard proteins (table) serve as MW markers during SDS-PAGE separations. Draw a hypothetical gel showing the migration pattern of these proteins given an optimal gel composition for complete separation.
b) What would the separation patterns look like if a lower than optimal % acrylamide was used, or a higher percentage?
c) Discuss the effects of post-translational modifications (phosphorylation, glycosylation) on the mass measurements of proteins by SDS-PAGE.
2.
a) In SDS-PAGE, some gels are prepared with a gradient. Which parameter is setup as a
gradient and how is this done?
b) Explain the effect of a gel gradient on the separation of low and high MW proteins.
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