Trouble Shooting on SDS-PAGE

Dr. Nishodh Saxena

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.

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

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.

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.

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.

• 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.

• Some of the proteins gets precipitate in lower concentration

forms the dark background.

• 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.

Protein bands fuzzy or poorly resolved

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.

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.

• 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.

Common Points that should be taken in Mind

• 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.

• 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

• 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.

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.

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.

• 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.

Skin proteins may be

present so always wear gloves starting from gel casting to gel staining.

Artifacts band observed at

approx. 67 kD in reduced

samples, especially with

silver staining

Excess reducing agent like

β mercaptoethanol or DTT.

Artifacts

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.

• 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.

Faint Staining

• 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.

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 leaked down the edges of gel, producing a localized frown at edge

Dye front curves down (frowns) at the edges

Electrode of the unit might be loosen results in running problem

• 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.

INTERESTING POINTS

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.

.

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.

THANKS

Quality is the responsibility of each and

every individual employee of the

organization.