2. Sodium Dodecyl Sulphate
PolyAcrylamide Gel Electrophoresis
(SDS-PAGE)
• It is an electropheritical technique based
on separations of the polypeptides by
the molecular mass.
• The nett charge carried by a protein is
depends on the binding of the SDS to a
single polypeptide independent of its size
- i.e.: the charge carried per unit mass
(or length, given proteins and nucleic
acids are linear macromolecules) of
molecule differs from protein to protein.
3. Brief of SDS-PAGE
1. Casting of gel
Basically it is a combination of 2 gels the top one is stacking gel
having the pH 6.8 with lower % of acrylamide and the bottom one is
separating (resolving) gel having the pH 8.8 with higher % of
acrylamide.
2. Sample preparation
Protein sample is prepared in sample buffer which contains SDS as well
as denaturing agent.
3. Loading of samples
4. Running the gel
5. Staining the gel
Either by Commassie stain or by Silver stain.
6. Drying the gel
4. Factors
There are many factors that affects
the quality of electrophoretic
separations it includes:
• Instrument assembly.
• Preparation of the gel, sample buffers and
Gel casting.
• The nature of the sample and its preparation.
• Run conditions.
5. Instrument Assembly
• Gel solution leaks out of sandwich
during casting.
• Plates and spacers are misaligned.
• Rubber gasket not seated properly.
• Glass plates are broken (chipped) at
edge.
6. Preparation of the gel, sample
buffers and Gel casting
• Minimize the time while
pouring the gel after
addition of APS and
TEMED and it should be
uniform.
• It is due to an unevenly
poured acrylamide
mixture.
• Here the lower half had
begun to polymerize
before the upper part was
poured.
7. • Some of the proteins gets precipitate in lower concentration
forms the dark background.
• As electrophoresis
proceeds the precipitates
re-dissolve and enter the
gel continuously, thus
forming a continuous
dark background of
unresolved polypeptides.
• Do not use sample buffer
which is precipitated, this
cause the same problem
and also alter the volume
of the final buffer to be
taken.
8. Protein bands fuzzy or poorly resolved
• Sample volume too high or
overloaded. Load sample in
less volume, as a tight layer in
the bottom of well. Height of
the stacking gel beneath the
well should be at least two
times the height of the sample
in the well.
Samples may be degrade keep
them on ice after boiling till
loading.
• Concentrate the samples if they
are diluted before loading
them.
9. Effect on electric field due to improper concentration
of the protein
• The electrical field was
affected by the non-uniform
concentration in the fourth
lane causing a narrowing of
the bands.
• A portion of the more
concentrated protein
precipitated during the
stacking process slight change
occurs while it is in
separating results in alteration
in the concentration.
• This gel runs too long results
in loss of dye front.
10. Streaking due to improper overlaying of separation
gel
• The separating gel is not
overlayed with butanol or
distilled water results in
rough top layer of
separating gel.
• Samples disproportionaly
stacks and precipitates. The
precipitates re-dissolved
gradually during the run,
forming streaks instead of
resolving into discrete
bands.
11. Common Points that should be taken in Mind
• The stacking gel length should be 1 cm from the well
bottom to the top of the separating gel for proper stacking
of the protein sample.
• Always use freshly prepared Ammonium Persulfate (APS).
• Sometimes artifacts found in the whole gel is due to glass
plates which is not properly washed or cleaned with
methanol or their might be the concentration of the β
mercaptoethanol or Dithiotheritol.
• Their should not be any bubble at the bottom and between
the well.
• Degassing the acrylamide solution leads to a more rapid
polymerization.
12. • Make sure the concentration of APS and TEMED is
correct.
• SDS concentration should not exceed 200 microgram / 30
microliter sample or it should not exceed 1.4gm / gm of
protein.
• Wiped all the glass plates, spacers, combs with the
methanol to remove the greasy materials.
• During protein sample treatment the sample should be
mixed by vortexing before and after the heating step for
best resolution.
• High salt concentration in sample or in buffers also causes
artifacts in the gel.
• Brief spin the samples before loading to remove any
aggregates.
13. • Gel cracking during polymerization
Make sure for no excess heat generation.
Use coolling reagents.
• Samples do not sink to bottom of well
Check the glycerol content in sample buffer.
Combs removed before stacking gel properly polymerized.
Let the stacking gel polymerize for 30 minutes to 1 hour.
• Poor sample wells
Distorted or broken wells are formed when the comb is not
removed carefully. Comb should be removed only in a
vertical manner.
Unreacted acrylamide continued to polymerize after comb
was removed. Rinse wells with distilled water immediately
after the comb is removed
14. • Detachment of slab gels from glass plates during gel
electrophoresis
Glass plates are not properly cleaned.
• Detachment of gel from the glass plates
Its due to unclean plates. Make sure plates are completely
dry after rinsing with distilled water.
• Bands on part of slab do not move down the gel
This is usually due to air bubbles between the plates
underneath the affected lanes. Make sure no bubbles are
present in the gel when pouring.
• More bands than expected observed for a purified protein
Minimize the time between sample preparation and
electrophoresis.
15. The nature of the sample
and its preparation
• Sample preparation yellow in color
Solution acidic – add NaOH until the solution turn
blue.
Too little bromophenol blue in sample buffer.
• Doublets observed where a single protein band is
expected on SDS-PAGE
Prepare fresh sample solution. Make sure the
concentration of β mercaptoethanol or
Dithiotheritol (DTT) is correct.
16. Samples diffused out of well
• Start electrophoresis
immediately after
loading samples.
• Diffusion of sample
out of the wells
vertically and
horizontally, before the
power was turned on.
Minimize the time
between sample
application and power
start – up.
17. • 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
• Insoluble material in the gel or
inconsistent pore size throughout
gel. Filter gel reagents before use
and ensure that the gel mixture is
well mixed before pouring the gel.
• Diffusion during migration through the stacking gel,
increase voltage by 25 % during stacking gel or make
sure the amount of APS and TEMED in the stacking gel
is proper.
18. Artifacts
Artifacts band observed at
approx. 67 kD in reduced
samples, especially with
silver staining
Excess reducing agent like
β mercaptoethanol or DTT.
Skin proteins may be
present so always wear
gloves starting from gel
casting to gel staining.
19. Gel was run with high current and overheated
This gel is run with
too much high
current in order to
complete the run
fast. Results in
overheating of gel
and bands are
completely
distorted.
20. Faint Staining
• The gel is kept in the water
after running instead of
fixing solution, than the gel
is stained.
• Here the small proteins are
diffused out of the gel.The
larger molecules stained
faint after the staining.
• Gel is required to place in
the fixing solution for the
precipitate the proteins to
prevent protein to diffused
out of the gel.
21. • Dye front curves up (smiles) at the edges
Gel is hotter in the middle than at the edges
Here the spacers act as heat sinks, lowering the
temperature at the edge of the gel.
22. Dye front curves down (frowns) at the edges
Air bubbles trapped
between glass plates at
bottom of gel sandwich can
be large enough to block
current flow and produce a
localized frown. Remove
air bubbles if present.
The spacers on the sides
were a bit loose, thus gel
next to the spacers was not
fully polymerized; current Electrode of the unit
leaked down the edges of might be loosen results in
gel, producing a localized running problem
frown at edge
23. INTERESTING POINTS
• Add 1X sample buffer to unused wells.
• Prepared samples (those which are boiled in sample buffer)
could be aliquoted and stored at – 20°C for 3 – 4 weeks or at
4°C for at least a week. Do not freeze-thaw repeatedly this
results in protein degradation. Before using these samples
they should be warmed at 37°C for a minute or 2 to
redisslove SDS which precipitates out of solution.
• If samples are prepared too early than it should be
immediately stored at 2 – 8°C to minimized further
denaturation.
• Always pour butylated water to overlay the separating gel
because if only water is added, due to surface tension the
edges of the gels gets curved and hence slight curving effects
found at the sides of the gels.
24. Skewed or distorted bands
Uneven gel interface.
The surface of the resolving gel
was uneven, so that when the
samples were stacked, the bands
started out with distorted shapes.
Uneven bands is also due to
uneven electric field.
• Poor polymerization around sample wells. Check the concentration of
TEMED.
• Excessive pressure applied to the gel plates when the gel is placed into
clamp assembly. Do not overtighten the screws on the clamp assembly.
• Use spirit level to make sure the gel apparatus is even. Overlay
separating gel with butylated water carefully.
25. BROKEN GELS
.
During drying if vacuum is released before gel is properly dried.
During staining improper
handling of gel or speed
of the shaker/rotator is
higher than required.
26. THANKS
Quality is the responsibility of each and
every individual employee of the
organization.
