SDS-PAGE异常电泳现象及分析
预备党员考察意见-元旦祝福
SDS-PAGE异常电泳现象及分析
（SDS-PAGE
“Hall of Shame”）
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You
may very well have prepared a nearly perfect gel,
and would have a difficult time
improving upon
the product. If that is so, then by all means
gloat about it! If you didn't do
such a hot
job don't despair. All good scientists learn from
their mistakes, in fact, without
making
mistakes most of us wouldn't learn much at all!
The
have run in past labs, with an example
or two from a research lab. They represent many of
the ways one can mess up a gel (but not all of
them - we're still finding new ways!). See
what features of your own gel(s) were
unsatisfactory - or at least less than perfect -
and
use the illustrations to figure out what
you might do to improve your technique.
To
critique your own work identify your symptoms and
use the gallery to select
appropriate
example(s). Each example is linked to a full sized
image with suggessted
explanations for the
symptoms. From this
what can be done to
correct the problem(s). If you wish you may browse
by descriptions of
symptoms, listed below the
gallery.
Symptoms 1—Smears
Smeared gels –
example 1
Smearing can have a variety of
causes, but most commonly it is due to an unevenly
poured acrylamide mixture or due to gross
overloading of protein.
In this
example the gel was not properly poured, so that
the lower half had begun to
polymerize before
the upper part was poured. The first gel mix began
to polymerize too
quickly. Rather than prepare
a new gel cassette, the students simply stopped
pouring,
prepared a new mix, and poured it on
top of the old. Obviously, the bond wasn't
particularly good.
Smeared gels – example
2
In this example a lot of protein was loaded
in each of the wells, all the way across. Most of
the lanes can still be interpreted, but
smearing is particularly evident in lanes 3 and 4.
Those lanes contained primarily one protein
(hemoglobin subunit), as did lanes 9 and 10.
However, the group that loaded 3 and 4
underestimated the protein concentrations in their
red cell lysate and red cell cytoplasm
fractions. Those fractions contained so much
protein
that samples had to be diluted prior
to preparing a protein assay tube. The students
forgot
to take the dilution factor into
consideration when determining protein
concentration.
The capacity of a mini-gel for
a mixed protein sample is 20 to 40 microgramswell,
depending on the resolution needed and number
individual polypeptides in the mix.
However,
if a pure protein is loaded, one single band will
contain all 20-40 micrograms,
and the result
is a mess.
Smeared gels –
example 3
Here is another example of gross
overloading, and it appears these students made
the
same mistake as in example 2. It looks as
though there was some inconsistency in the gel
also.
Smeared gels – example 4
Smearing can be a normal consequence of
running membrane-associated proteins with a
high lipid content in a discontinuous gel. In
discontinuous PAGE, sample proteins are
super-
concentrated by a combination of two factors.
First, the rate of migration is suddenly
slowed as the sample moves from a non-
restrictive stacking gel to a separating gel that
restricts movement. Second (and more
important), a pH change affects the
electrophoretic mobility of proteins in
the sample, causing the sample to check up
abruptly.
A sample of a half cm or so in
height becomes compressed into a layer of bands a
few
micrometers thick, and the local
concentration of protein goes up considerably.
Membrane-associated proteins tend to precipate
at lower concentrations, thus lanes
containing
such proteins often have a dark band of
precipitated proteins at the very top.
As
electrophoresis proceeds the precipates re-
dissolve and enter the gel continuously,
thus
forming a continuous dark background of unresolved
polypeptides. Many of the
membrane samples
shown on these gels will show that characteristic.
The resolved bands in the membrane
protein lanes (4 and 7) appear to consist of
similar
amounts of protein, however in lane 4
the capacity of the gel for at least some of the
membrane proteins was exceeded, resulting in a
dark smear.
Symptoms 2—Streaks
Streaks –
example 1
This one isn't so bad. The lane with
the streaks (left of lane 5) is quite usable, in
fact. The
streaks came about because a portion
of the more concentrated proteins in the
membrane fraction precipated during the
stacking process, then returned to solution after
a fairly short time. Because of slight
imperfections in the separating gel surface (it
may
have been allowed to dry a little before
adding the stacking gel), the protein
concentration
was not uniform across the lane,
and streaks rather than a uniform darker
background are
the result.
Streaks –
example 2
The fourth lane is
streaked due to the effect described above. The
electrical field was
affected by the non-
uniform concentration in the fourth lane (the dark
one with streaks),,
causing a narrowing of the
bands. Note that the dye front was lost in this
gel - it was
allowed to run too long.
Streaks – example 3
This one kind of
stinks. Evidently, this group did not use an
overlay after pouring the first
gel layer. The
result was a separating gel with the very rough
top that you see here. As
samples were stacked
they were disproportionately concentrated in the
'valleys,' where
the proteins precipitated.
The precipitates re-dissolved gradually during the
run, forming
streaks instead of
resolving into discrete bands.
Symptoms
3—Bands too light
Light bands – example 1
It looks like this problem was caused by badly
formed wells andor careless loading of
sample
onto the gel. The background is dark and uneven,
suggesting that protein was in
the running
buffer itself. Most likely the apparatus was
jarred and much of the samples
was slopped out
of the wells. The spilled sample would not resolve
into bands,since it
continuously
entered the gel from the upper buffer compartment.
Note the presence of a horizontal line,
continuous along several lanes (arrow). That would
suggest that the wells were too shallow, and
some material spilled into adjacent wells.
This symptom also shows up in perfectly good
gels when the electrodes are hooked up
backwards for a few minutes. Protein migrates
out of the wells instead of into the stacking
gel. Catching the problem within a few minutes
salvages some of the information, but the
pH
effect that causes efficient stacking is
compromised, sample is lost, and protein
contaminates the running buffer.
Light
bands – example 2
Judging from the lack of
other symptoms, it appears that the gel simply
didn't stain well.
Coomassie blue dye staining
solution can become contaminated with SDS if it is
recycled.
The dye becomes less effective and
proteins don't show up as well as with fresh dye.
As
long as the proteins were precipitated in
the gel by the acidified alcohol in the stain
solution, the problem could be
corrected simply by re-staining with fresh dye.
Light bands – example 3
The 'X' marks
the lane next to lane 5 where the amount of
protein was overestimated in
the original
sample, the sample was prepared to the wrong
concentration for
electrophoresis, or too
little was loaded - perhaps sample wasn't properly
drawn up into
the syringe. The absence of any
major bands, compared to the other samples,
suggests
that the well was simply
underloaded.
Light bands – example 4
This is one of the more disappointing symptoms
you might encounter. Let's say you were
extremely meticulous,did everything perfectly,
and ran your gel so that the dye front was
perfectly even and right at the bottom. You
rinsed the gel in deionized water, then left it on
the shaker. Being late in the day, you forgot
to replace the water with the stain. It was
stained the next day, but here's the result:
Notice that the smaller proteins
diffused rather quickly out of the gel, while the
larger ones
at the top diffused much more
slowly, and took the stain. The acidified alcohol
in the stain
solution and in the destain
solutions is essential, as it precipates proteins,
preventing them
from diffusing out of the
acrylamide matrix.
Symptoms 4—Missing Dye
Front
Partial dye front – example 1
After
doing so much work getting the gel set up, it is a
shame to be late taking it down.
Relative
mobilities cannot be determined with accuracy if
the dye front is partially off of the
gel.
In this case the gel was
internally calibrated - that is, molecular weight
standards were
included on the gel. An
arbitrary reference point can be used for setting
up the standard
curve, since the lanes were
quite even.
No dye front – example 2
This
one was allowed to run until the dye front
completely ran off. Since the lanes are
somewhat distorted,relative mobility
measurement will be compromised. With an intact
dye front, lanes could be compared even if the
gel was distorted or of different height on
one side or the other. Relative migration is
the same for a gel of uniform composition, no
matter what the actual height of the
resolving gel.
No dye front – example 3
The curvature of this gel suggests that the
spacers on the sides were a bit loose, causing
a field effect near the edges (a 'frowning'
gel). It will be difficult to get accurate
relative
mobility measurements because the dye
front was allowed to run off. With an intact dye
front as a reference point, lanes could be
compared with one another despite the 'frown.'
Symptoms 5—Bands only at top of
gel
Stopped too soon – example 1
It is
obvious where the dye front was on this gel. There
was room to continue
electrophoresis, which
would have spread out the bands even more and
improved
resolution. Note that the pattern is
consistent with the other gels that
Relative
mobility, for gels of identical composition, is
the same for identical proteins. The
resolution is improved with the distance the
proteins are permitted to migrate, until
resolution becomes limited by diffusion.
Stopped too soon – example 2
Each of these
gels – one of about 7%T and one about 14%T – was
looking good.
Someone became impatient and
stopped the run early. They are usable, but the
separation would have been much better if the
dye front had been allowed to reach the
bottom
of the gel. This will happen when you run late,
the instructor gets hungry, and
you
aren't there to keep him from taking down your
gel.
Symptoms 6—Miscellaneous
Failed Gels - Crooked, Broken, Smeared, Weird
Crooked bands
Examples of this problem are
scattered throughout the 'hall of shame.' The
surface of the
resolving gel was uneven, so
that when the samples were stacked, the bands
started out
with distorted shapes.
Crooked bands also result from an uneven
electric field, but usually that type of
distortion
is symmetrical.
Bad wells
If samples run over into adjacent wells, you
will see what looks like a continuous protein
band running across the wells (arrow). In
fact, that's exactly what it is. This is usually
what
happens when the wells are too short, or
perhaps sample is accidentally spilled outside
the wells.
In addition, we may have used
old sample buffer. An advantage of using Cleland's
reagent (dithithreitol, or DTT) to reduce
disulfide bonds is that it doesn't stink as badly
as
2-mercaptoethanol. However, the latter
compound is much more stable. Disulfide bonds
link individual polypeptides together and
maintain some proteins in a folded state under
otherwise denaturing conditions. This
causes them to run unpredictably.
Dense
Gel
...or should it be dense investigators?
The gel mix was clearly incorrect. The acrylamide
concentration was so high that only the
smallest proteins entered the gel to any extent.
The bands were distorted due to overloading
and the fact that beyond a reasonable
concentration the most concentrated
gels tend not to polymerize evenly.
Broken Gel
This can be really frustrating.
You did a very nice job, then the gel broke during
handling.
Keep in mind that the gel will still
be usable as long as you save the pieces.
Field Effects
To some extent this
is normal, although for some reason the field
effect was exaggerated
here. The spacers
interrupt the electric field at the edges of the
gel, so that the 'pull' (or
'push' if you
prefer) is biased to one side the closer the
sample is to the edge of the gel.
The result
is the 'frowning'effect you see here.
The
individual bands sometimes smile, but if the gels
have any expression at all, they
always frown.
I guess you would too if we zapped you with
electricity and drowned you in
blue dye
Symptoms 7—Failed Gels with Multiple Symptoms
Multiple symptoms – example 1
Many times the gels you run will have a
variety of symptoms. Since the object of a
critique
is to improve your work, it is
important to determine what caused the undesirable
effects.
Then you can take steps to improve
the quality of your gels.
This one has no dye front - electrophoresis
was terminated too late. Bands are crooked -
the top of the resolving gel was uneven, most
likely due to failure to use a properly overlay.
On the right side, samples were overloaded.
Multiple symptoms – example 2
This gel
shows some field effects (curvature - lanes are
not straight). There is some
overloading, and some bands are crooked
due to an uneven resolving gel surface.
Multiple symptoms – example 3
Uneven
loading (different amounts in adjacent wells) has
caused some compression in
the standards lane.
There are tears in the gel and discoloration
throughout. This indicates
that it was not
properly handled - handling a gel with bare hands
leaves protein on the
surface that
stains with Coomassie. Sometimes you get very nice
fingerprints.
Symptoms 8—Bizarre Results
Explained
Bizarre – example 1
The large
container of glycine looked the same as the large
container of choline chloride.
Choline is a
lot more hydroscopic than glycine, which should
have been a clue that the
wrong component was
used in the electrode buffer. Choline is a basic
compound and
certainly does not substitute for
glycine. We caught the error and replaced the
buffer after
electrophoresis had
proceeded for some time, but it was too late for
this one.
Bizarre – example 2
This
one was kindly contributed by T. Sedlacek, Czech
Republic. It was run with high
current
and overheated
Bizarre – example 3
Here are two gels that were contributed by E.
Morales Rayas. A likely explanation is that
each time there was a delay between loading
the samples and actually running the gel.
The
middle lanes of the first gel show an alternating
pattern in which every other lane is
either
wide or compressed. Bands on the second gel spread
out toward the edges.
When samples sit in a
well the proteins begin to diffuse into the
stacking gel, both
vertically and laterally.
Smaller proteins diffuse more rapidly than do
larger ones. If
proteins in a sample diffuse
laterally they may alter the electric field
affecting adjacent
lanes, especially if the
samples in adjacent wells contain predominantly
higher mass
polypeptides. The problem isn't so
bad when all of the samples are of similar
composition
(second gel) and the lanes
are loaded in sequence. However distortion still
occurs.
Bizarre – example 4
Evidently it is not a good idea to store tris-
glycine electrode buffer in a gallon plastic
bottle
on a shelf in the didn't see any mold
in the buffer, but by golly it was there, all
right.
Notice that in each example the
gel below the dye front was cleared following
destaining.
The persistent blue background is
from mold proteins that continued to penetrate the
gel
throughout the procedure.