7 Electrophoresis
Objectives:
A) To perform agarose gel electrophoresis of the proteins isolated in last
week's experiment and B) to interpret the banding patterns produced by
these proteins.
Introduction: Electrophoresis is the migration of charged molecules in an electric field.
It is an important analytical technique for the separation of biomolecules
(proteins, amino acids, nucleic acids, lipids). The electrophoretic mobility
of a charged particle is the extent of movement of that particle in an
electric field. Electrophoretic mobility of small molecules is greater than
that of large molecules with the same charge density. In a medium of
constant viscosity and voltage, the mobility of a particle is directly
proportional to its charge to size ratio:
µ proportional to Q / r
where- µ = electrophoretic mobility
Q = net charge of the molecule
r = radius (size)
If the molecular size of the molecules in a sample is approximately the
same, the electrophoretic mobility is directly proportional to the net
charge:
µ
proportional to
Q
Usually, the pH of the gel is high enough (often above pH 8) so that nearly
all proteins have net negative charges and move toward the anode when
the current is switched on. Molecules of similar size and charge will
migrate through the gel as a single band.
In gel electrophoresis, a semi-solid gel (matrix) is used as the supporting
medium. The two gels of choice for separating proteins or nucleic acids,
namely agarose and polyacrylamide, are both cross-linked polymers.
Thus, both gels are porous in composition. The advantage gained with
this porosity is that pore size can be controlled to yield gel matrices with a
wide variety of uniform pore sizes.
An agarose gel is a semi-solid medium that represents a polysaccharide
obtained from agar. It is composed of a linear polymer of D-galactose that
is cross-linked to galactose 3.6 anhydro L-galactose. Agarose gels can
provide very large pore sizes. Because of the large pore sizes, agarose is
the medium of choice when analyzing nucleic acids, the largest
biomacromolecules in size.
Additionally, it is the preferred
electrophoretic medium when one analyzes the structure of proteins or
protein complexes in their native conformation.
Agarose gel
electrophoresis of a protein is commonly used to determine a) its
isoelectric point or b) whether it has quaternary structure.
Polyacrylamide gel electrophoresis (PAGE) is the method of choice
when one examines denatured proteins and/or nucleic acids, or relatively
small biomacromolecules such as peptides and DNA fragments. A
polyacrylamide gel consists of a polymer of acrylamide
[CH2=CH−CONH2] that is occasionally cross-linked with bis-acrylamide
(N,N'-methylene
bis-acrylamide)
[CH2=CH−CONH−CH2−NHCO−CH=CH2]. TEMED (N, N, N', N'tetramethylene diamine) and ammonium persulfate are needed to initiate
the polymerization of acrylamide and bis-acrylamide. The pore size is
determined by the total acrylamide concentration. Very small pore sizes
can be obtained with polyacrylamide gels by varying the concentration of
acrylamide (the bis-acrylamide concentration usually remains constant).
The degree of resolution of a mixture of molecules will depend on, to a
large extent, the concentration of the matrix. Consequently, increasing the
percent (w:v or v:v) of the matrix, either acrylamide or agarose, enhances
the separation or resolution of small molecules. On the other hand, low
percent matrix gels increases the resolution of larger molecules.
Common Electrophoresis Patterns
1
2
3
4
5
-
6
Direction
of
migration
*
+
Lane 1 is the pattern indicative of a well-resolved mixture of proteins
except for (*). The “band” indicated by the (*) actually represents
several unresolved proteins. The “smile” in Lane 2 is indicative of a
sample that was electrophoresed too quickly. The “frown” indicated
in Lane 3 indicates uneven heating of the gel during electrophoresis.
Lane 4 represents the pattern observed when the protein sample is
overloaded (i.e., too much protein was loaded). Lane 5 indicates a
degraded protein sample. Lane 6 represents a pure protein sample.
Note: The banding pattern exemplified in the above figure will be
observed regardless of the electrophoretic medium.
Denaturing PAGE is generally performed if, for example, one wants to
ascertain the relative molecular mass of a protein or its quaternary
structure. In denaturing PAGE, the detergent sodium dodecyl sulfate
(SDS) is used as the denaturant. This type of electrophoresis is generally
referred to as SDS-PAGE. When SDS-PAGE analysis is used to
determine the relative molecular mass of a protein, one must compare the
migration of the protein under analysis to that of protein standards. The
relationship between migration distance and molecular mass is linear
when analyzed via a semi-log plot:
Logarithmic Relationship between the
Molecular Mass of a Protein and Its
SDS-PAGE Electrophoretic Mobility
Molecular mass (kD)
80
70
60
50
40
30
20
10
0.2
0.4
0.6
0.8
1.0
Relative mobility
The location of a protein that is electrophoresed through a gel can be
ascertained with the aid of a dye. The most common method of
visualization is by staining with a solution containing Coomasie Brilliant
Blue, the same dye used in the Bradford reagent. After excess dye is
removed by washing the gel in a destain solution, the proteins appear as
bands in the gel matrix. Coomasie staining very easily detects microgram
quantities of protein loaded into a gel. A much more sensitive form of
protein detection relies on silver. Silver staining, although much more
expensive than Coomasie staining, is a necessity when the protein of
interest is present at sub-microgram quantities. Coomasie staining is done
with both agarose and polyacrylamide gels, whereas as silvers staining is
reserved for the latter.
Capillary electrophoresis is an analytical electrophoretic technique used
when a very small quantity of sample is available. In this method,
electrophoresis is carried out in very thin capillary tubes. The use of these
thin tubes facilitates rapid heat dissipation. Consequently, very high
electric fields are used resulting in both high resolution and separation
times of minutes rather than hours.
Paper electrophoresis is the most popular procedure used to fractionate
amino acids and peptides. Low molecular weight proteins are not
separated by paper electrophoresis because the cellulose matrix limits their
resolution. Cellulose acetate electrophoresis of proteins is the method of
choice in clinical laboratories that test for sickle cell anemia.
In today’s experiment, we are going to electrophorese casein and BSA,
the proteins upon which you conducted ammonium sulfate fractionation,
using polyacrylamide as the gel matrix. The gel will be stained, and then
destained. The migration of these proteins will be compared to a mixture
of standards (indicated in the following table) and their respective
molecular weights estimated.
Bio-Rad Kaleidoscope prestained Standards
Protein
Color*
Size
Myosin
Blue
216,000
β Galactosidase
Magenta
132,00
BSA
Green
78,000
Carbonic Anhydrase
Violet
45,700
Soybean trypsin inhibitor
Orange
32,500
Lysozyme
Red
18,400
Aprotinin
Blue
7,600
Procedures:
A) SDS PAGE
1. An 8% SDS PAGE gel has already been prepared. For reference purposes the
“recipe” is listed below:
Solution components
Resolving (or separating) Stacking gel
gel
H2O
2.3 mL
1.4 mL
30% Acrylamide
1.3 mL
0.33 mL
1.5 M Tris pH 8.8
1.3 mL
---
1.0 M Tris pH 6.8
---
0.25 mL
10% SDS
0.05 mL
0.02 mL
10%
persulfate
ammonium 0.05 mL
0.02 mL
TEMED
0.003 mL
0.001 mL
2. SDS PAGE is a denaturing gel. The proteins must be boiled in the presence of
SDS to denature them. Thus we need to add 5 µL of Laemmli loading dye
solution. This contains a dye, used for determining how long to let a gel run.
The dye is smaller than the proteins, thus runs faster. As long as the dye has not
run off of the gel we can be sure we have not lost the protein. The Laemmli also
contains a reducing agent to reduce the disulfide bonds.
3. Boil the samples, molecular weight marker as well, for 4 minutes.
4. Remove from the boiling water and place on ice.
5. Load the gels into the apparatus. The short plates need to be on the inside.
6. Fill the apparatus with 1 X SDS running buffer; fill up to the top of the tall glass
plate, inside and outside.
7. Load the samples, any order is fine AS LONG AS YOU RECORD THE
ORDER IN YOUR NOTEBOOK. People usually load the markers on the left.
Loading the sample can be difficult. Place the pipette tip into the buffer and
touch the back plate. Slowly raise the tip until you feel the tip move forward to
the tall plate. Place the tip over the well and SLOWLY pipette out the sample.
The sample should sink to the bottom of the well.
8. Once all the samples, and markers are loaded start the gel at 100 volts. After 20
minutes increase the voltage to 150 volts.
9. After the gel has run for about 1 hour turn off the power supply and remove the
gel. Carefully, using a spatula or razor blade pry the glass plates apart. This is
done by inserting the tip of the blade between the plates and using a GENTLE
twisting action.
10. The gel will stick to one plate, immerse the plate and the gel in the running
buffer and carefully remove the gel from the glass. The gel has a tendency to
stick to the plate and rip. T
11. Cut one corner of the gel so you know the orientation, I usually cut the lower
left hand corner, the side with markers, which I usually run on the left. There is
nothing magical about this orientation. What is vitally important is that YOU
know what sample was loaded where.
12. Place the gel in staining solution and gently shake. Staining should take about
15 minutes.
13. After 15 minutes, pour the stain in the bottle labeled “Used Stain”.
14. Rinse the gel with deionized water to remove the staining solution.
15. Place gel in de-stain solution and shake.
16. Once the de-stain solution gets too blue remove the blue de-stain and replace it
with fresh de-stain.
17.
a.
Determine the distance each protein band in the standard lane migrated
from the well by using a ruler to measure from the bottom of the well to the
center of the band. Record the distance in cm.
b.
Use Microsoft Excel ® to plot log MW of the standards versus the distance
migrated.
c.
Using linear regression analysis, determine the molecular weight of both
casein and globulin by plugging their migration distances into the generated
equation.
A plot of pI vs. distance yields a
line with a negative slope. Why?
On your image, you must identify of the sample loaded into the well. Your report
must also include a discussion of the sample’s integrity based on the shape of the
band (i.e., narrow, broad, smile, frown, smear, single band, multiple bands, etc.), and
the sample’s estimated molecular weight. Does this experimentally determined
molecular weight match the estimate given on page 42?
Make a photocopy of the gel or sketch it in your notebook:
One partner needs to turn in the actual gel with your lab report, the other partner
needs to turn in a photo copy of the gel.
Electrophoresis STUDY GUIDE
1. What is electrophoresis?
2. In a medium of constant viscosity and voltage, what determines the electrophoretic
mobility of a substance?
3. When is the electrophoretic mobility of molecules in a sample directly proportional
to their net charges?
4. What determines pore size of a polyacrylamide gel?
5. Explain, in detail, how you would prepare 40 mL of 1% (w:v) agarose gel for an
electrophoresis experiment.
6. A sample composed of a mixture of proteins − myoglobin, β-lactoglobulin B,
cytochrome c, and bovine carbonic anhydrase − yielded one of the two patterns
diagrammed below after agarose gel electrophoresis at pH 8.3 and stained with
Coomasie. The pI of each protein is indicated in the following table
Protein
pI
Carbonic anhydrase (bovine)
6.5
Cytochrome c
9.6
β-Lactoglobulin
5.10
Myoglobin
7.00
a. Identify the correct electrophoretic pattern (A or B).
b. Identify each protein.
Hint: To solve this problem, use the equation Q = pI - pH.
A.
B.
2 proteins with same
net charge
-
The well
+
-
The well
+
7.
Why do most proteins migrate towards the positive electrode?
8. Suppose you have 10ng of your favorite protein. What type of electrophoretic
analysis should you run if you need to determine the relative Mr of your protein
sample? Why?
9. Why is the detergent SDS a denaturant and not a degradative agent?
10. You have loaded 2ng of your protein into an agarose gel to determine its pI. Upon
completion of electrophoresis followed by staining with Coomasie for an hour and
overnight destaining with constant agitation, only the standards were visible. Why?