Electrophoresis is a simple, rapid, and highly sensitive analytical technique to study the properties of proteins and nucleic acids, and has become a principle tool in analytical chemistry, biochemistry, and molecular biology. Polyacrylamide gel electrophoresis (PAGE) can be used to analyze the size, amount, purity, and isoelectric point of polypeptides and proteins. Sodium dodecyl sulfate polyacrylamide discontinuous gel electrophoresis (SDS PAGE) is the most commonly used system whereby proteins become separated strictly by their size, but there are different variations of this technique.

1. www.expedeon.com
INTRODUCTION
Electrophoresis is a simple, rapid, and highly sensitive analytical technique to study the properties of proteins and nucleic acids, and has become a
principle tool in analytical chemistry, biochemistry, and molecular biology. Polyacrylamide gel electrophoresis (PAGE) can be used to analyze the
size, amount, purity, and isoelectric point of polypeptides and proteins. Sodium dodecyl sulfate polyacrylamide discontinuous gel electrophoresis
(SDS PAGE) is the most commonly used system whereby proteins become separated strictly by their size, but there are different variations of this
technique.
Separation of molecules is dependent upon the gel pore size of the support matrix used. The two types of support matrices commonly used in gel
electrophoresis, polyacrylamide and agarose, act as porous media and behave like a molecular sieve. Polyacrylamide has a small pore size and is
ideal for separating most proteins and smaller nucleic acids. The way that molecules separate is also dependent on the conditions under which an
electrophoretic run takes place.
Detailed below are some common SDS PAGE problems, along with their probable cause and potential solution:
Sample Preparation Issues
Problem Cause Solution
Yellowing of sample / load
buffer
 pH of sample / load buffer is too
acidic, and turns bromophenol from
blue to yellow
 Reverse by adding Tris base until sample buffer turns back
to normal blue color
Sample is very thick and
sticky / gluey
 The sample contains a high amount
of DNA or carbohydrate
 Ultrasonic DNA fragmentation during cell lysis and protein
solubilization (PPS Silent® Surfactant / NVoy Stabil-PAC)
 Addition of endonucleases (e.g. BaseMuncher
Endonuclease)
 Precipitate protein with TCA / sodium deoxycholate or
acetone to reduce carbohydrate content
Sample Loading Issues
Problem Cause Solution
Sample does not stay in
the well
 Density of the sample / load buffer is
lower compared with the running
buffer
 Check that glycerol / sucrose has been added to sample /
load buffer
 Use appropriate sample / load buffer for the gel type
(RunBlue™ LDS Sample Buffer – TEO-Tricine, RunBlue™ 4x
LDS Sample Buffer – Bis-Tris)
Tracking dye does not
enter the gel
 No current / poor connection
 Reversed polarity of electrodes
 A lack of bubbles forming on electrodes indicates a poor / no
connection – check electrical supply
 If electrical supply is normal, then check the electrodes to
see if they have been put in the wrong positions
Protein bands in unloaded
wells
 Overloading of sample wells into
empty adjacent wells
 Poor polymerization of stacking gel,
giving rise to partial well formation
and leakage
 Running buffer is contaminated
 Sample on outside of pipette tip flows
across gel, especially when fingers
between wells do not reach or extend
past top of the front plate
 Protein bands in unloaded wells will be the same size as the
sample wells
 Protein staining will be seen in all tracks, repeat with a new
precast gel
 Replace with fresh running buffer
 Use gels with fully formed fingers and/or wipe off outside of
pipette tip before adding sample to the well
Unexpected bands from
loaded wells
 Contamination of sample / load buffer
 Leakage from / overflowing wells
 Replace with fresh sample / load buffer (RunBlue™ LDS
Sample Buffer – TEO-Tricine, RunBlue™ 4x LDS Sample
Buffer – Bis-Tris)
 Check integrity of sample Wells
 Avoid overloading wells
SDS PAGE Electrophoresis
Troubleshooting Tips
TECHNOTE

2. www.expedeon.com
Sample Preparation Issues
Problem Cause Solution
High / uneven background
staining
 Samples with a high degree of
proteolysis
 Overloaded wells
 Sample / load buffer impurities /
contamination
 Dirty equipment
 Impure reagents
 Too long in stain
 Use protease inhibitor in sample buffer (Proteoloc™)
 Do not overload wells, but use the suggested volume /
concentrate the sample
 Use new sample / buffer Use appropriate load buffer
for the gel type (RunBlue™ LDS Sample Buffer – TEO-
Tricine, RunBlue™ 4x LDS Sample Buffer – Bis-Tris)
 Ensure clean equipment is used
 Ensure use of high purity water and reagents
 Restrict length of time in stain, wash gel in water or
destain for a minimum of 30 mins
Staining is uneven
 Gel not shaken / agitated
sufficiently during staining
 Increase agitation and / or time during staining
Protein Separation Issues
Problem Cause Solution
No bands observed
 Gel does not contain protein
 Imager not working / incorrect
parameters used to visualize gel
 Use alternative stain to confirm presence of protein
 Refer to instrument manual to troubleshoot / contact
imager manufacturer
Poor resolution
 Impure reagents
 Overloading of sample
 Incorrect buffer pH
 Incorrect running buffer
 Incorrect sample / load buffer
 High temperature
 Ensure use of high purity water and reagents
 Do not overload sample
 Adjust buffer pH
 Ensure that the running buffer is correct one for gel buffer
system (RunBlue™ SDS Running Buffer, RunBlue™ MES
Run Buffer, RunBlue™ MOPS Run Buffer)
 Use appropriate sample / load buffer for the gel type
(RunBlue™ LDS Sample Buffer – TEO-Tricine, RunBlue™
4x LDS Sample Buffer – Bis-Tris)
 Reduce the current to slow down the gel run, reduce
temperature and obtain an even gel temperature
Distorted / fuzzy bands
 Temperature gradient from center of gel
to gel surface
 Sample has a high salt content
 Poor polymerization, bubbles or insoluble
materials in the gel
 Insufficient reducing agent / sample not
reduced
 Ensure proper cooling of gel to reduce temperature
gradient
 Desalt sample
 Repeat with a new gel
 Revise concentration of reducing agent / use β-
mercaptoethanol (β-ME) or dithiothreitol (DTT) (RunBlue™
DTT Reducer) and heat sample (70–80°C for 10 minutes)
Protein does not enter the
resolving gel / streaking
of protein in the gel
 Protein aggregation / precipitation
 Dissolution of the precipitate during the
electrophoretic run
 When running both reduced and non-
reduced samples on the same gel, lateral
diffusion of reducing agent during the run
causing reduction during the run
 Centrifuge samples following denaturation to remove
insoluble materials
 Decrease the amount of sample loaded
 Dilute sample preparation and use a continuous buffer
system
 Keep one or two empty lanes between reduced and non-
reduced samples
Protein dimer or double
bands in gel
 Due to disulf
 ide bonds
 Sample not sufficiently reduced or not
reduced
 Use fresh sample / load buffer (RunBlue™ LDS Sample
Buffer – TEO-Tricine, RunBlue™ 4x LDS Sample Buffer –
Bis-Tris)
 Ensure that the sample has been heated with a reducing
agent such as β-mercaptoethanol (β-ME) or dithiothreitol
(DTT) (RunBlue™ DTT Reducer)
Electrophoretic Run Issues
Problem Cause Solution
Run time is too short
 Gel buffer may be too dilute
 Running buffer may be too
concentrated
 Use gel buffer that is more concentrated
 Dilute the running buffer (RunBlue™ SDS Running Buffer,
RunBlue™ MES Run Buffer, RunBlue™ MOPS Run Buffer)
Gel shrinkage  Gel has become dehydrated  Rehydrate gel in water
Run time is too long
 Running buffer may be too dilute
 Gel buffer may be too concentrated
 Use running buffer that is more concentrated
 Dilute the gel buffer
Leaking from
electrophoresis tank
 Improper assembly of tank
 Damage to tank
 Assemble correctly according to manufacturer’s
instructions
 Check for damage to the tank

3. www.expedeon.com
RELATED PRODUCTS FROM EXPEDEON:
Expedeon produce a range of products to assist you with your SDS PAGE electrophoresis. Please click here to view our extensive range.
References:
 Gel Electrophoresis of Proteins – A Practical Approach. 3rd edition. BD. Hames. Series ed. Oxford, United Kingdom: Oxford University Press.
http://www.oup.co.uk/PAS