Electrophoresis is the migration of charged molecules in response to an electric
field. Their rate of migration depends on the strength of the field; on the net
charge, size and shape of the molecules, and also on the ionic strength, viscosity,
and temperature of the medium in which the molecules are moving. As an
analytical tool, electrophoresis is simple, rapid, and highly sensitive. It is used
analytically to study the properties of a single charged species, and as a
There are a variety of electrophoretic techniques, which yield different
information and have different uses. Generally, the samples are run in a support
matrix, the most commonly used being agarose and polyacrylamide. These are
porous gels, and under appropriate conditions, they provide a means of
separating molecules by size. We will focus on those methods used for proteins.
These can be denaturing or nondenaturing. Nondenaturing methods allow
recovery of active proteins and can be used to analyze enzyme activity or any
other analysis that requires a native protein structure. Two commonly used
techniques in biochemistry are sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). SDS-PAGE separates
proteins according to molecular weight and IEF separates according to isoelectric
point. This laboratory exercise will introduce you to SDS-PAGE.
The gel matrix used is a crosslinked acrylamide polymer. This electrophoretic
method separates the proteins according to size (and not charge) due to the
presence of SDS. The dodecyl sulfate ions bind to the peptide backbone, both
denaturing the proteins and giving them a uniform negative charge.
The gels we will be running use a discontinuous system, meaning that they
have 2 parts. One is the separating gel, which has a high concentration of
acrylamide and acts as a molecular sieve to separate the proteins according to
size. Before reaching this gel, the proteins migrate through a stacking gel, which
serves to compress the proteins into a narrow band so they all enter the
separating gel at about the same time. The narrow starting band increases the
resolution. This part of the gel has a lower concentration of acrylamide to avoid
a sieving effect.
The stacking effect is due to the glycine in the buffer, the low pH in the
stacking gel, and the higher pH in the running buffer. At the low pH, the
glycine has little negative charge, and thus moves slowly. The chloride ions
move quickly and a localized voltage gradient develops between the 2. As the
gel runs, the low pH of the stacking gel buffer is replaced by the higher pH in
the running buffer. This maintains a discontinuity in the pH and keeps the
glycine moving forward (any glycine molecules behind would acquire a higher
charge and speed up). Since there is no real sieving going on, the proteins
(which have intermediate mobility) form a tight band, in order of size, between
the slower glycine and the faster chloride ions. The separating gel buffer has
a higher pH, so the glycine molecules become more negatively charged and
move past the proteins, and the voltage gradient becomes uniform. The proteins
slow down in the smaller pore size of the separating gel and separate according
: You will be given protein molecular weight standards, several
different solutions containing individual proteins, and a sample of the same
serum you used in the protein quantitation lab. Your job is to determine the
molecular weights of the individual proteins and the major components in the
serum sample. You will run each sample on 2 gels, one you prepare yourself
and a commercial precast gel, and compare the results.
Before doing electrophoresis, you must know the amount of protein in each
sample. Determine the protein concentrations of each of your samples using a
protein assay before coming to the lab to do any electrophoresis. For this
exercise, the only sample of unknown protein concentration is the serum that
you used for one of your unknowns last week. The amount of protein to be
loaded depends on the thickness and length of the gel, and the staining system
to be used. Using the Coomassie Blue staining system, as little as 0.1 mg can
be detected, but more will be easier to see. As a guide, use 0.5–5 mg for pure
samples (one or very few proteins) and 20–60 mg for complex mixtures where
the protein will be distributed amongst many protein bands. Overloading will
decrease the resolution.
The apparatuses used in gel casting or running electrophoresis
vary; make sure you look over the appropriate manuals before you operate.
Unpolymerized Acrylamide is a Neurotoxin. Be Careful! Do not pour
unpolymerized acrylamide down the sink, wait for it to polymerize and dispose
of it in the trash.
TEMED (N, N, N', N'-tetramethylethelenediamine) is also not very good for
you and is very smelly; avoid breathing it. Open the bottle only as long as
necessary, or use it in the hood.
Make sure gel plates are clean and dry. Do not get your fingerprints on
them or the acrylamide will not polymerize properly.
Prepare gel solutions (separating and stacking), but do not add polymerizing
agents, APS and TEMED (this would start the polymerization).
Lay the comb on the unnotched plate and mark (on the outside, using a
Sharpie) about 1 cm below the bottom of the teeth. This will be the level
of the separating gel. If available, use an alumina (opaque, white) plate, for
the notched plate, as this conducts heat away from the gel more efficiently
than glass. Set up the gel plates, spacers, and plastic pouch in the gel
casting as described in the manufacturer’s directions. When everything is
completely ready, add TEMED to the separating gel solution, mix well, and
pour it between the plates, up to the mark. Wear gloves if you pour directly
from the beaker. You can also use a disposable pipette. Work quickly or
the solution will polymerize too soon. Carefully layer isopropanol (or
water-saturated butanol) on top of acrylamide so it will polymerize with
a flat top surface (i.e., no meniscus). Do this at the side and avoid large
drops, so as not to disturb the gel surface. When the leftover acrylamide
in the beaker is polymerized, the acrylamide between the plates will also
If you are running the gel on the same day, prepare samples while the
acrylamide is polymerizing. Otherwise, wait until you are ready to run the
(i) You will need a sample of each unknown substance, plus the molecular
weight standards. Prepare samples in screw-cap microcentrifuge tubes.
The protein content should be at 1–50 mg in 20–30 mL sample.
The total sample volume that can be loaded depends on the thickness
of the gel and the diameter of the comb teeth. For Genei apparatuses,
this is ~ 30 mL/well. To prepare the sample, mix 7–10 mL of the sample
(depending on protein concentration) +20 mL 2X sample buffer containing
10% b-mercapto-ethanol (BME). Use the BME in the hood - it stinks!
For dilute samples, mix 40 mL of the sample and 10 mL 5X sample buffer
and add 2 mL of BME. Heat to 90°C for 3 minutes to completely denature
proteins. It is important to heat samples immediately after the
addition of the sample buffer. Partially denatured proteins are much
more susceptible to proteolysis and proteases are not the first proteins
to get denatured. (Heat samples to 37°C to redissolve SDS before running
the gel if samples have been stored after preparation).
(ii) If you want the proteins in the sample to retain disulfide bonds, do not
add BME. If both reduced and nonreduced samples will be run on the
same gel, leave at least 3–4 empty wells between samples, since the
BME will diffuse between wells and reduce proteins in adjacent samples.
(iii) MW Stds: 7 mL of Rainbow stds +10 mL of sample buffer (do not make
in advance). Heat to 37°C before use.
After the separating gel has polymerized, drain off the isopropanol. Add
TEMED to the stacking gel solution, pour the solution between the plates,
and insert the comb to make wells for loading samples. The person putting
in the comb should wear gloves. Keep an eye on this while it’s polymerizing
and add more gel solution if the level falls (as it usually does), or the wells
will be too small.
After polymerization, do not cut the bag; we reuse them. The gel may be
stored at this point by taping the bag shut to prevent drying.
When ready to run the gel: mark the position of each well, since they are
difficult to see when full.
Remove comb and rinse wells with running buffer. See the manual
directions for setting up the gels in the buffer chambers. The apparatus can
run 2 gels simultaneously. There is a blank plate to use when running
only one. Fill the upper chamber with running buffer first and check for
leaks. Adjust the plates if necessary. Load the samples using a micropipettor
with gel-loading tips (these are longer and thinner than the normal tips).
This will be demonstrated. Do not load samples in the end wells. Make
sure to write down which sample was loaded in each well.
Electrophoresis (takes 1–2 hours).
Connect the gel apparatus to the power supply and run at 15 mA/gel
until the tracking dye (blue) moves past the end of the stacking gel. Increase
the current to 20–25 mA/gel but make sure the voltage does not get above
210 V. Run until the blue tracking dye moves to the bottom of the separating
For the BioRad apparatus, do not exceed 30 mA, regardless of the number
Disassemble the apparatus and carefully separate the gel plates using a
Cut off the stacking gel and any gel below the blue tracking dye. Note the
color of each of the molecular weight standards, as they will all be blue
after staining. Wash 3X with distilled water.
Place the gel in a plastic staining container and add Coomassie Blue
staining solution. Keep it in this 1 hour overnight. Wash again with water.
You can wrap the gel in plastic wrap and Xerox or scan it to have a copy.
The gel may also be dried.
Measure the length of the gel (since you cut off the bottom, this is the distance
traveled by the dye).
Measure the distance traveled by each of the molecular weight standards.
Measure the distances of each unknown band.
For samples lanes with many bands (serum in this exercise), measure all
bands in those with just a few and the major bands in those that have many.
Prepare a standard curve by plotting log MW versus relative mobility (Rf,
distance traveled by protein divided by distance traveled by dye). Use this and
the mobility of bands from your fractions to determine the MW of the unknown
proteins. (Review standard curves from the protein quantitation lab if necessary.)
MW of proteins that do not run very far into the gel or run near the dye front
will not be accurate.
If you have reduced and unreduced samples, compare the number of bands
and MW of each to determine the number of subunits.
When adding SDS, avoid making too much foam, which makes measuring
and pouring difficult.
Separating gel: (15 mL, enough for two gels) 10% acrylamide.
40% Acrylamide/bisacrylamide mix 3.55 mL.
1.5 M tris pH 8.8, 3.75 mL, H2O 7.4 mL, 10% SDS 150 mL, 10% ammonium
persulfate (APS) 150 mL (prepared fresh), TEMED 6 mL.
Stacking gel: (5 mL) 5% acrylamide.
Compresses the protein sample into a narrow band for better resolution.
40% Acrylamide/bisacrylamide mix 0.625 mL.
0.5 M tris pH 6.8, 1.25 mL, H2O 3.0 mL, 10% SDS 50 mL, 10% APS 50 mL,
TEMED 5 mL.
2X sample buffer (10 mL)—store in the freezer for an extended time.
SDS must be at room temperature to dissolve
H2O 1.5 mL, 0.5 M Tris pH 6.8, 2.5 mL, 10% SDS (optional) 4.0 mL,
glycerol 2.0 mL, BPB 0.01%, b-mercaptoethanol (optional) 0.1 mL.
30 g Tris Base, 144 g glycine, dissolve in sufficient H2O to make 1.5 L and
put into final container.
Add 1.5 g SDS (Caution: do not inhale dust).
Final pH should be around 8.3, but do not adjust it or the ionic strength
will be too high and the gel will not run properly. If the pH is way off, it was
made incorrectly or is old and has some contamination.
The running buffer can also be made more concentrated (5X or 10X) and
diluted as needed to save bottle space.