Electrophoresis Discussion Examples | How to Interpret Band Position, Intensity, and Molecular Weight

What Is Electrophoresis?

Electrophoresis is a method in which charged molecules are moved through a gel by applying an electric field and separated according to differences in molecular size and charge. DNA is negatively charged because it contains phosphate groups and therefore migrates toward the positive electrode in an agarose gel. Proteins differ in charge and shape in their native states, but in SDS-PAGE, SDS gives them an almost uniform negative charge, allowing them to be separated mainly according to differences in molecular weight.

The gel acts like a molecular sieve. Smaller molecules pass through the pores of the gel more easily and migrate farther. Larger molecules migrate less easily and remain closer to the wells. These differences in migration distance are used to estimate the size of the target molecules.

Main Items to Include in the Results

In electrophoresis results, organize the sample name, type of gel, gel concentration, electrophoresis conditions, molecular weight marker, band positions, band intensities, extra bands, and presence or absence of smearing. In a report, rather than simply attaching a photograph, it is important to clearly state what was loaded into each lane and how each band was interpreted.

Main Items to Include in the Results

• Name of the sample used
• Type of gel
• Gel concentration
• Electrophoresis buffer
• Electrophoresis voltage
• Electrophoresis time
• Staining method
• Type of molecular weight marker
• Sample loaded into each lane
• Position of the target band
• Estimated molecular weight or DNA fragment size
• Band intensity
• Presence or absence of extra bands
• Presence or absence of smearing
• Validity of the results
• Sources of error and points for improvement

Reference Experimental Values and Band Analysis Examples for Electrophoresis

Here, band positions, migration distances, band intensities, molecular weight estimation, and concentration estimation obtained in electrophoresis experiments are organized as reference experimental values that are easy to discuss in reports.

In electrophoresis, molecules such as DNA, RNA, and proteins migrate in an electric field. Because migration distance varies depending on molecular size, charge, gel concentration, and electrophoresis conditions, the molecular weight or fragment size of an unknown sample can be estimated by comparison with a molecular weight marker.

Reference Experimental Conditions

Example Bands in DNA Agarose Gel Electrophoresis

The following is a reference example in which a DNA marker and unknown DNA samples were electrophoresed on a 1.0% agarose gel. Migration distances were measured downward from the origin.

In DNA electrophoresis, smaller fragments generally migrate faster through the gel and appear farther from the origin. The approximate size of an unknown sample can be estimated by comparing its migration distance with that of the marker.

Relationship Between Migration Distance and Molecular Weight

In electrophoresis, migration distance and molecular weight themselves do not necessarily have a simple proportional relationship. For DNA fragments and proteins in SDS-PAGE, the logarithm of molecular weight may show an approximately linear relationship with migration distance.

By plotting migration distance on the horizontal axis and log10 size on the vertical axis to prepare a calibration curve, the size of an unknown DNA fragment can be estimated from its migration distance.

Example Estimation of Unknown DNA Size Using a Calibration Curve

Suppose the calibration curve obtained from the DNA marker is approximated as follows.

log10(DNA size) = −0.278 × Migration distance + 3.98

If the migration distance of the unknown DNA is 3.5 cm,

log10(DNA size) = −0.278 × 3.5 + 3.98 = 3.007

DNA size = 10^3.007 = approximately 1020 bp

Therefore, this unknown DNA is estimated to be a fragment of approximately 1000 bp.

Example Estimation of Protein Molecular Weight by SDS-PAGE

In SDS-PAGE, SDS binds to proteins and gives them an almost uniform negative charge, so they are separated mainly according to differences in molecular weight.

In SDS-PAGE, proteins with smaller molecular weights migrate farther through the gel. A single band may indicate relatively high purity, whereas multiple bands may indicate the presence of impurities or degradation products.

Example Calculation of Relative Mobility Rf

In electrophoresis, the migration distance of a band relative to the distance traveled by the dye front or electrophoresis front may be expressed as relative mobility.

Relative mobility Rf = Migration distance of the band ÷ Migration distance of the dye front

If the dye front migrated 6.0 cm and the band of Unknown Protein A migrated 2.6 cm,

Rf = 2.6 ÷ 6.0 = 0.433

Using Rf makes comparison with standard markers easier even if the electrophoresis distance differs slightly among experiments.

Relationship Between Rf and Molecular Weight of Protein Markers

The larger the Rf value, the smaller the molecular weight of the protein tends to be. When preparing a calibration curve, the relationship between Rf and log10 molecular weight is used.

Example Estimation of Concentration From Band Intensity

Band intensity in electrophoresis provides an indication of the amount of DNA or protein in the sample. Here, a reference example is shown in which the amount of DNA is roughly estimated from DNA band intensity.

If the band intensity of the unknown sample is similar to that of the 50 ng standard, the amount of DNA electrophoresed can be roughly estimated as approximately 50 ng. However, intensity and amount may not be perfectly proportional because of uneven staining or saturation.

Example Quantification of Band Intensity

The following is a reference example in which band intensity was quantified using image-analysis software.

Because the integrated intensity of the unknown sample, 46740, is close to the 45000 of the 50 ng standard, the amount of the unknown sample is estimated to be approximately 50 ng.

Interpretation When Multiple Bands Are Observed

Interpretation When Smearing Is Observed

In electrophoresis, a broad, spread-out signal rather than a clear band is called a smear.

When a smear is present, unlike a clear band representing a single molecular weight, molecules of various sizes may be mixed together.

Differences in Separation Depending on Gel Concentration

The higher the gel concentration, the finer the pore structure becomes, making it suitable for separating smaller molecules. On the other hand, larger molecules migrate less easily.

If a gel concentration unsuitable for the size of the target fragment is used, band separation may become poor or the migration distance may become too short.

Effects of Electrophoresis Time and Voltage

High voltage can shorten electrophoresis time, but may cause heating of the gel and band smearing. To obtain bands that are easy to analyze, the voltage and electrophoresis time must be set appropriately.

Appearance of Bands Depending on Loading Amount

Although stronger band intensity generally indicates a larger amount of sample, overloading may saturate the staining and make it difficult to compare quantities accurately from band intensity.

Typical Examples of Electrophoresis Failure

Example of How to Write the Results

Agarose gel electrophoresis using a DNA marker showed bands at 5000 bp, 3000 bp, 1500 bp, 1000 bp, and 500 bp. DNA with larger molecular sizes had shorter migration distances, while smaller DNA migrated farther from the origin. In Unknown DNA A, a single band was observed 3.5 cm from the origin, corresponding to the position of the 1000 bp marker. Therefore, the size of Unknown DNA A was estimated to be approximately 1000 bp.

Using the calibration curve prepared from the marker, the log10 size corresponding to a migration distance of 3.5 cm for Unknown DNA A was approximately 3.01. Converting this value to DNA size gave approximately 1020 bp, which agreed well with the visual estimate of approximately 1000 bp obtained by comparison with the marker. Therefore, the size estimation using the calibration curve is considered reasonable.

In Unknown DNA B, two bands corresponding to approximately 1500 bp and approximately 500 bp were observed. This indicates that the sample contained multiple DNA fragments. In contrast, Unknown DNA C showed a smear throughout the lane. This suggests that the DNA may have degraded into fragments of various sizes or that an excessive amount of sample was loaded.

Points for Connecting the Results to the Discussion

In a discussion of electrophoresis, it is important to explain not only the presence or absence of bands but also their position, intensity, shape, comparison with markers, and experimental conditions in relation to one another.

• Have the band positions of the molecular weight marker and unknown sample been compared?
• Can the molecular weight or DNA size of the unknown sample be estimated from migration distance or Rf?
• Can a calibration curve be prepared using the relationship between the logarithm of molecular weight and migration distance?
• Can the meanings of a single band, multiple bands, smearing, and no band be distinguished?
• Can sample amount or concentration be roughly estimated from band intensity?
• Can it be explained that overloading causes band smearing and reduces quantitative accuracy?
• Can the effect of gel concentration on the range of sizes that can be separated be discussed?
• Can the effects of voltage and electrophoresis time on migration distance and band shape be explained?
• Can causes of abnormal band patterns such as smiling, smearing, and material remaining in the wells be considered?
• Can the validity of the experiment be judged together with the results of negative controls, positive controls, and markers?

Example Discussion

In this experiment, electrophoresis was used to estimate the band positions and molecular sizes of unknown samples. In the DNA marker, the 5000 bp band was observed near the origin, whereas the 500 bp band was observed farther away. This is because smaller DNA fragments pass more easily through the pores of the agarose gel and migrate faster in an electric field.

Unknown DNA A showed a single band at the same position as the 1000 bp marker. In addition, using a calibration curve prepared from the marker, the DNA size corresponding to a migration distance of 3.5 cm was calculated to be approximately 1020 bp. Therefore, Unknown DNA A is considered to contain a DNA fragment of approximately 1000 bp as its main component. Because only a single band was observed, the major DNA fragments in the sample were considered relatively uniform.

Unknown DNA B showed two bands corresponding to approximately 1500 bp and approximately 500 bp. This result indicates that two types of DNA fragments were present in the sample. If the sample was a PCR product, nonspecific amplification may have occurred, while if it was a restriction-enzyme-treated sample, the bands may represent generated fragments. When bands other than the target band are observed, the reaction conditions and whether purification was performed should be checked.

In contrast, Unknown DNA C showed a smear rather than a clear band. Smearing tends to occur when a sample has degraded into fragments of various sizes or when too much sample has been loaded. Samples with high salt concentrations or containing impurities may also show blurred bands. Therefore, when a smear is observed, the storage condition, concentration, purification state, and loading amount of the sample should be reviewed.

Possible sources of error include errors in reading the band center position, variations in gel concentration, differences in electrophoresis time and voltage, and heating of the gel. In particular, electrophoresis at high voltage may heat the gel and produce smile-shaped bands in which migration distances differ between the center and edges. Therefore, for molecular-weight estimation, it is important to compare the sample with a marker on the same gel and keep the electrophoresis conditions constant.

Summary

In electrophoresis, the DNA size or protein molecular weight of an unknown sample can be estimated by comparing band positions with a molecular weight marker. Band intensity provides an indication of sample amount, while the number and shape of bands provide clues for judging purity, impurities, degradation, and overloading.

This reference example covered DNA agarose gel electrophoresis, SDS-PAGE, migration distance, Rf, calibration curves, band intensity, multiple bands, smearing, gel concentration, electrophoresis conditions, and examples of failure. In a report, it is useful to organize band position, intensity, and shape as numerical values or observations and discuss them in relation to molecular weight markers and experimental conditions.

How to Interpret Band Position

Band position in electrophoresis is important information for estimating molecular size. In general, smaller DNA fragments and lower-molecular-weight proteins migrate faster through the gel and appear farther from the wells. Larger molecules have more difficulty passing through the gel pores and therefore remain closer to the wells.

When interpreting band position, always compare it with a molecular weight marker or size marker. By comparing the sample band position with bands of known size in the marker, the approximate molecular weight or number of base pairs of the molecules in the sample can be estimated. In addition to visual comparison, measuring migration distance and preparing a standard curve enables more quantitative evaluation.

How to Interpret Band Intensity

Band intensity reflects the amount of DNA or protein contained in a sample. In general, under the same staining conditions, a darker band indicates a larger amount of the target substance. Conversely, a faint band may indicate a small sample amount, low extraction yield, low amplification efficiency, insufficient staining, or similar factors.

However, band intensity is not a perfectly quantitative value. It is also affected by loading amount, staining conditions, exposure conditions, gel thickness, electrophoresis time, and sample diffusion. When comparing band intensities, the samples must be compared on the same gel and under the same staining and imaging conditions.

Role of the Molecular Weight Marker

A molecular weight marker is a mixture of DNA fragments or proteins of known size. In electrophoresis, the molecular size of a sample is estimated by comparing its band position with the marker. Without a marker, even if bands appear, it becomes difficult to determine their size.

In DNA electrophoresis, sizes are read in bp or kb, while in protein electrophoresis they are read in kDa. Because different markers contain different size ranges, a marker suitable for the size of the target substance must be used.

How to Estimate Molecular Weight

To estimate molecular weight more accurately, the migration distance from the well is measured for each band in the molecular weight marker. In DNA electrophoresis and SDS-PAGE, there is a range in which an approximately linear relationship is observed between the logarithm of molecular size and migration distance. Therefore, a standard curve is prepared from the marker migration distances and sizes, and the sample band migration distance is fitted to the curve to determine its size.

However, a standard curve can only be used when the marker and sample were electrophoresed on the same gel under the same conditions. Because migration distance changes when gel concentration or electrophoresis conditions differ, it is not appropriate to use a standard curve obtained from another gel.

Estimate the size of a sample band using the relationship between the logarithm of molecular size and migration distance.

Discussion of DNA Electrophoresis

DNA electrophoresis uses the negative charge of the phosphate groups in DNA to move DNA fragments toward the positive electrode. In an agarose gel, smaller DNA fragments pass through the pores more easily and migrate farther. It is commonly used to confirm PCR products, restriction-enzyme digestion products, and extracted DNA.

In a discussion of DNA electrophoresis, check whether a band of the target size is present, whether extra bands or smearing are present, and whether the band intensity is sufficient. For PCR products, the presence of a band of the target size is important. For extracted DNA, little degradation is desirable. For restriction-enzyme-treated samples, the expected number and sizes of bands are important.

Discussion of SDS-PAGE

SDS-PAGE is an electrophoresis method used to separate proteins mainly according to differences in molecular weight. SDS denatures proteins and gives them an almost uniform negative charge. Therefore, lower-molecular-weight proteins migrate faster through the gel, while higher-molecular-weight proteins remain closer to the wells.

In a discussion of SDS-PAGE, check whether the target protein band is near the expected molecular weight, the band intensity, the presence or absence of extra bands, the degree of purification, and the presence or absence of degradation products. If many bands other than the target band are present, the sample may not have been sufficiently purified.

Discussion When the Target Band Appears

If a band is observed near the target size or expected molecular weight, the target molecule may be present in the sample. For PCR products, the target sequence may have been amplified, while for protein samples, the target protein may have been expressed or purified.

However, the presence of a band at the same position does not completely prove that it is the target substance. Nonspecific PCR products or impurity proteins with similar molecular weights may appear at the same position. If necessary, confirmation by restriction-enzyme treatment, sequencing, Western blotting, mass spectrometry, or similar methods is required.

Discussion When No Band Appears

If the target band does not appear, possible causes include absence of the target substance in the sample, a small amount of the target, failure of PCR or another reaction, insufficient protein expression, insufficient sample loading, or insufficient staining. In DNA electrophoresis, DNA degradation, the presence of PCR inhibitors, or inappropriate primer conditions may also be involved.

In SDS-PAGE, possible causes include a small amount of protein, protein precipitation, degradation, loss during sample preparation, or insufficient staining sensitivity. When no band appears, the possible causes should be organized step by step from sample preparation through electrophoresis and staining.

Discussion When the Band Is Faint

If a band is faint, the amount of the target substance may be small. In DNA experiments, possible causes include low PCR amplification yield, low DNA extraction yield, low sample loading amount, or weak staining. In protein experiments, possible causes include low expression levels, loss during purification, low sample amount, or insufficient staining sensitivity.

However, a faint band does not necessarily mean that the target substance is absent. The target may be present in a small amount, or it may simply appear faint because of the imaging conditions.

Discussion When the Band Is Too Dark

If a band is extremely dark, possible explanations include a large sample amount, a large amount of the target substance, strong PCR amplification, or high protein expression. However, loading too much sample may make the band thick or blurred and make accurate judgment of position and amount difficult.

An excessively dark band may also affect adjacent lanes or cause smearing. For quantitative comparisons, the sample should be diluted, the loading amount reduced, or the imaging conditions adjusted.

Discussion When Extra Bands Appear

If bands other than the target band appear, the sample may contain molecules other than the target substance. In DNA electrophoresis, possible causes include nonspecific PCR products, primer dimers, undigested DNA, partially digested products, or RNA contamination. In SDS-PAGE, possible causes include impurity proteins, degradation products, unpurified components, or multiple subunits.

The properties of impurities can be estimated from the position and intensity of the extra bands. Bands on the lower-molecular-weight side of the target band may be degradation products or small nonspecific products, while bands on the higher-molecular-weight side may represent nondenatured complexes or undigested fragments.

Discussion When Smearing Occurs

Smearing is a condition in which a band appears spread across the gel rather than as a clearly defined line. In DNA experiments, smearing occurs when DNA has been degraded or sheared and fragments of various lengths are mixed together. In protein experiments, smearing may result from degradation, excessive loading, the effects of salts or impurities, or insufficient denaturation.

Smearing may indicate reduced sample quality or inappropriate electrophoresis conditions. However, high-molecular-weight DNA such as genomic DNA readily shows smearing when sheared during extraction or handling.

Discussion When Bands Curve or Become Distorted

If bands are curved or distorted rather than straight, possible causes include poor gel preparation, irregular well shape, high salt concentration in the sample, excessive loading, heating during electrophoresis, or excessively high voltage. Excessive sample amounts in adjacent lanes may also affect migration in surrounding lanes.

Band distortion reduces the accuracy of molecular-weight estimation from migration distance. In particular, when using a standard curve, it is important that the bands migrate in a straight and uniform manner.

Discussion of Primer Dimers

In electrophoresis of PCR products, a very small band may appear on the low-molecular-weight side. This may represent a primer dimer. Primer dimers are short DNA fragments amplified when primers bind to one another.

Causes of primer-dimer formation include problems with primer design, an annealing temperature that is too low, a high primer concentration, and a low amount of template DNA. If the target band is faint and the primer-dimer band is strong, optimization of PCR conditions is required.

Discussion of Nonspecific Bands

If bands other than the target size appear in PCR products, nonspecific amplification may have occurred. Extra bands are produced when primers bind not only to the target sequence but also to other DNA sequences, causing different DNA fragments to be amplified. Possible causes include an annealing temperature that is too low, inappropriate primer design, high Mg²⁺ concentration, or too many cycles.

If nonspecific bands appear, the PCR conditions must be reviewed. If a nonspecific band is darker than the target band, side reactions may have proceeded more strongly than the intended reaction.

Discussion of Protein Degradation Products

If multiple bands appear on the lower-molecular-weight side of the target protein in SDS-PAGE, they may represent degradation products of the protein. Proteins may be degraded by protease activity or inappropriate storage conditions. When degradation occurs, the protein migrates as fragments smaller than its original molecular weight.

Methods for preventing degradation include working at low temperature, processing samples within a short time, and using protease inhibitors. However, the actual method should follow the instructions in the laboratory manual.

Effect of Gel Concentration

Gel concentration greatly affects molecular separation. In agarose gels, low-concentration gels are suitable for separating large DNA fragments, while high-concentration gels are suitable for separating small DNA fragments. In polyacrylamide gels, higher gel concentrations are also more suitable for separating smaller proteins and smaller nucleic acids.

If the gel concentration does not match the size of the target molecule, the bands may be too close together or separation may be insufficient. It is important to select a gel concentration appropriate for the size of the target substance.

Effects of Electrophoresis Voltage and Time

If the electrophoresis voltage is too high, molecules migrate quickly, but heating may distort the gel or broaden the bands. If the voltage is too low, electrophoresis takes longer and the bands may diffuse. If the electrophoresis time is too short, separation may be insufficient, while if it is too long, small molecules may migrate out of the gel.

In electrophoresis, it is important to stop the run under conditions in which molecules of the target size have migrated to an appropriate position and can be easily compared with the marker. Inappropriate electrophoresis conditions cause errors in evaluating band position and intensity.

Effect of the Electrophoresis Buffer

The electrophoresis buffer maintains the pH and ionic strength during electrophoresis and allows electric current to flow. If the type or concentration of buffer is inappropriate, the migration speed of molecules and band shape may be affected. Using old buffer or buffer prepared with an incorrect dilution may make electrophoresis unstable.

Buffers such as TAE and TBE are used in DNA electrophoresis, while electrophoresis buffers containing SDS are used for protein electrophoresis. It is important to prepare the buffer specified in the laboratory manual correctly.

Effect of Sample Loading Amount

If the sample loading amount is too small, the band becomes faint and difficult to detect. If too much sample is loaded, the band may become too dark or thick, and smearing or lane distortion may occur. When quantitative comparison is desired, it is important to load the same amount of sample into each lane.

If DNA or protein concentrations differ among samples, loading the same volume does not mean that the same amount is being compared. When necessary, concentrations should be standardized or loading amounts adjusted.

Discussion of Loading Errors

In electrophoresis, samples must be loaded correctly into the wells. If sample leaks out of the well, enters an adjacent well, damages the well, or contains air bubbles, bands may become weak or distorted or contamination may occur between adjacent lanes. Particularly with small wells, differences in loading technique can greatly affect the results.

Effects of Staining and Destaining

Bands after electrophoresis are visualized using DNA stains or protein stains. If staining is insufficient, the bands appear faint, while excessive destaining may weaken the signal. Conversely, if the background is too strong, faint bands become difficult to distinguish.

Even for the same sample, appearance varies depending on staining time, the condition of the staining solution, and imaging conditions. When comparing band intensity, it is important to keep staining and imaging conditions consistent.

Discussion When the Molecular Weight Differs From the Expected Value

If the target band position differs from the expected size, several causes can be considered. For DNA, possible causes include nonspecific amplification, incomplete restriction-enzyme digestion, differences in plasmid conformation, or RNA contamination. For proteins, possible causes include post-translational modification, degradation, dimer formation, insufficient reduction, insufficient denaturation, or abnormal electrophoretic mobility of the protein.

Particularly for proteins, the actual molecular weight and the apparent molecular weight on SDS-PAGE may not completely agree. This is because migration can be affected by amino acid composition, modifications, and structure.

Discussion of Negative and Positive Controls

Negative and positive controls are important in electrophoresis used to confirm PCR products or protein expression. If a band appears in the negative control, contamination or nonspecific reactions are suspected. If no band appears in the positive control, there may have been problems with the PCR conditions, reagents, enzymes, electrophoresis, or staining operations.

If the control experiments give appropriate results, the reliability of the target-sample results increases. Conversely, if the control experiments do not give the expected results, the target-sample results must also be interpreted carefully.

When the Result Can Be Considered Good

Electrophoresis results can be considered good when the molecular weight marker is clearly separated, a clear band is present near the target size, and there are few extra bands or smears. It is also important that the control experiments give the expected results and that migration in each lane is not distorted.

Confirmation of the target substance requires a comprehensive judgment based not only on band position but also on band intensity, extra bands, sample purity, and control-experiment results. Even when only one clear band is visible, additional confirmation may be necessary when appropriate.

Example Discussion When the Experiment Did Not Go Well

If electrophoresis does not go well, possible causes are considered from results such as no bands, faint bands, smearing, many extra bands, failure of the marker to separate, or distorted bands. It is easier to organize the discussion by considering sample preparation, gel concentration, electrophoresis conditions, sample loading amount, staining conditions, molecular weight marker, and control experiments separately.

How to Write Points for Improvement

In a discussion of electrophoresis, including not only sources of error but also points for improvement makes the report easier to organize. Improvements are easier to organize by dividing them into sample preparation, gel preparation, electrophoresis conditions, sample loading, and staining and imaging.

Improvements to Sample Preparation

• Check the concentration of DNA or protein
• Prevent sample degradation
• Remove impurities and salts as much as possible
• Mix thoroughly with sample buffer
• Adjust the sample amount when necessary

Improvements to Electrophoresis Conditions

• Select a gel concentration appropriate for the target size
• Prepare the electrophoresis buffer correctly
• Do not set the voltage too high
• Use an appropriate electrophoresis time
• Always include a molecular weight marker

Improvements to Loading and Staining

• Load samples without damaging the wells
• Avoid contamination of adjacent lanes
• Use the same loading amount for each lane
• Use appropriate staining and destaining times
• Keep imaging conditions consistent
• Repeat electrophoresis when necessary to confirm reproducibility

Difference Between a Superficial Discussion and a Good Discussion

In a discussion of electrophoresis, simply writing that “a band appeared,” “it was dark,” or “it was faint” results in a superficial discussion. Relating band position, molecular weight marker, molecular size, band intensity, extra bands, smearing, and electrophoresis conditions produces a more persuasive discussion.

Examples of Expressions That Can Be Used in Reports

The following expressions can be used when writing the results and discussion of electrophoresis. Adjust the necessary parts according to your own experimental results.

• A band was confirmed near the target size by comparison with the molecular weight marker.
• Smaller molecules migrate more easily through the gel and appear farther from the wells.
• The molecular size of the sample can be roughly estimated from band position.
• Band intensity reflects the amount of target molecule in the sample but is also affected by loading amount and staining conditions.
• Possible causes of the faint target band include insufficient sample amount, insufficient extraction yield, and reduced reaction efficiency.
• Extra bands may be derived from nonspecific products, impurities, or degradation products.
• The presence of smearing suggests that the sample may have been degraded or sheared.
• If the gel concentration is not appropriate for the size of the target molecule, separation becomes insufficient.
• If the electrophoresis voltage is too high, heating may distort the bands.
• Obtaining the expected results in control experiments is important when interpreting the target-sample results.

Points to Check When Discussing Electrophoresis

Checking the following points before writing the report makes the discussion easier to write.

• Have you clearly stated what was loaded into each lane?
• Have you compared the sample with a molecular weight marker?
• Have you explained the position of the target band?
• Have you discussed the intensity of the target band?
• Have you considered the causes of extra bands?
• Have you checked for the presence or absence of smearing?
• Have you distinguished the principles of electrophoresis for DNA and proteins?
• Have you considered whether the gel concentration was appropriate for the target size?
• Have you discussed the effects of electrophoresis voltage and time?
• Have you considered sample loading amount and loading errors?
• Have you considered the effects of staining and imaging conditions?
• Do the points for improvement correspond to the sources of error?

Summary

Electrophoresis is an experiment used to separate DNA and proteins according to differences in size and charge. In DNA electrophoresis, negatively charged DNA migrates toward the positive electrode, and smaller fragments migrate farther. In SDS-PAGE, SDS gives proteins an almost uniform negative charge and separates them mainly according to differences in molecular weight.

Band position is used to estimate molecular weight or DNA fragment size, while band intensity provides an indication of the amount of target molecule in the sample. However, because band intensity is also affected by loading amount, staining conditions, and imaging conditions, caution is required for strict quantification. Extra bands may indicate nonspecific products, impurities, or degradation products, while smearing may indicate degradation, overloading, or poor electrophoresis conditions.

In a report, rather than simply writing that “a band appeared,” discuss the molecular weight marker, band position, intensity, extra bands, smearing, gel concentration, electrophoresis conditions, and sample loading amount in relation to one another. Electrophoresis results provide important clues not only to the presence or absence of the target substance but also to the amount, purity, and degradation state of the sample.