Examples of Discussion for Biochemistry Lab Reports | How to Write About Enzyme, Protein, and DNA Experiments

Important Perspectives for Discussion in Biochemistry Laboratory Reports

In biochemistry experiments, the results are greatly affected by the condition of biomolecules.
For enzymes, activity affects the results; for proteins, concentration and denaturation; and for DNA, purity and the presence or absence of degradation.
Therefore, in the discussion, it is necessary to consider not only the measured values but also sample handling, temperature, pH, reaction time, reagent concentration, and losses during operations.

In addition, biochemistry experiments often use absorbance, fluorescence, electrophoresis bands, enzyme reaction rates, and standard curves to judge the results.
In a report, it is important to clearly state what the measured values mean and on what basis the target substance was confirmed.

Main Items to Include in the Results

In the results of biochemistry experiments, measured values, standard curves, concentrations, activities, band patterns, molecular-weight estimates, yields, purity, and similar data are organized.
The items to include differ depending on the type of experiment, but it is important to summarize the results so that it is clear which conclusions were drawn from which measured values.

Main Items to Include in the Results

• Name of the sample used in the experiment
• Measurement conditions
• Measured values such as absorbance, fluorescence, and color development
• Standard curve or calibration curve
• Protein concentration
• Enzyme activity
• Specific activity
• Reaction rate
• DNA concentration
• DNA purity
• Position and intensity of electrophoresis bands
• Comparison with molecular weight markers
• Comparison with theoretical or expected values
• Sources of error and points for improvement

Basics of Discussing Enzyme Experiments

Enzyme experiments investigate the rate at which an enzyme converts a substrate into a product and changes in activity caused by reaction conditions.
Enzyme activity is affected by temperature, pH, substrate concentration, enzyme concentration, inhibitors, reaction time, and other factors.
Therefore, in the discussion, explain under which conditions the reaction rate or absorbance change became larger or smaller.

Because enzymes are proteins, their three-dimensional structures may change at high temperatures or extreme pH values, resulting in decreased activity.
In a discussion of an enzyme experiment, it is useful to relate changes in enzyme activity to the three-dimensional structure of the enzyme and the condition of the active site.

How to Determine Enzyme Activity and Discuss It

Enzyme activity represents how much substrate is consumed or how much product is formed per unit time.
When changes in absorbance are used, the reaction rate is determined from the change in absorbance over time and, when necessary, converted into an amount of substance using a molar absorption coefficient or calibration curve.

When determining the reaction rate, the linear portion at the beginning of the reaction is often used.
As the reaction proceeds, the substrate concentration may decrease and products may accumulate, causing the reaction rate to become nonconstant.

When Absorbance Is Used in Enzyme Reactions

In enzyme reactions, if the product or substrate absorbs at a specific wavelength, the progress of the reaction can be monitored from changes in absorbance.
If the absorbance increases, the amount of product that absorbs at that wavelength may be increasing, while if the absorbance decreases, the substrate may be being consumed.

When absorbance is used, the measurement wavelength, blank correction, turbidity of the reaction solution, bubbles, contamination of the cuvette, and linearity of the measurement range are important.
If the absorbance is too high or too low, the accuracy of conversion to concentration may decrease.

Effect of Temperature on Enzyme Activity

Enzyme activity is strongly affected by temperature.
As the temperature rises, molecular motion becomes more active and the frequency of collisions between enzyme and substrate increases, so the reaction rate increases within a certain range.
However, if the temperature becomes too high, the enzyme protein denatures and the three-dimensional structure and active site collapse, causing activity to decrease.

Therefore, enzymes have an optimum temperature at which activity is highest.
In a temperature-activity graph, activity may increase from low temperature to the optimum temperature and then decrease sharply at higher temperatures.

Effect of pH on Enzyme Activity

Enzyme activity is also greatly affected by pH.
Acidic and basic amino acid residues are involved in enzyme active sites, and their charge states change depending on pH.
If the pH is not appropriate, the charge states required for substrate binding and the reaction change, causing enzyme activity to decrease.

In addition, under extreme pH conditions, the three-dimensional structure of the enzyme protein may also change.
Therefore, enzymes have an optimum pH at which activity is highest.

Discussion of Substrate Concentration and Enzyme Reaction Rate

In the low-substrate-concentration range, increasing the substrate concentration makes encounters between enzyme and substrate more likely and increases the reaction rate.
However, when the substrate concentration becomes sufficiently high, the enzyme’s active sites are almost completely occupied by substrate and the reaction rate approaches its maximum value.
Under this condition, adding more substrate does not greatly increase the reaction rate.

This type of relationship may be discussed as a Michaelis-Menten-type enzyme reaction.
In a report, it is useful to explain in which concentration range the reaction rate increased and where it reached a plateau.

Causes of Low Enzyme Activity

Causes of enzyme activity being lower than expected include enzyme inactivation, inappropriate temperature conditions, pH deviations, insufficient substrate concentration, deviations in reaction time, contamination by inhibitory substances, and poor sample-storage conditions.
Because enzymes are proteins, their activity may decrease because of heating, freeze-thaw cycles, prolonged standing, strong acids or bases, organic solvents, and similar factors.

Discussion of Protein Quantification Experiments

In protein quantification experiments, methods such as the Bradford assay, Lowry method, BCA assay, and UV absorption method are used to determine the protein concentration in a sample.
In many cases, a calibration curve is prepared using a standard protein of known concentration, and the concentration of an unknown sample is determined from its absorbance.

In a discussion of protein quantification, consider the linearity of the calibration curve, differences in reactivity between the standard protein and the unknown sample, the absorbance measurement range, interfering substances in the sample, and errors in pipetting.

Discussion of Protein Calibration Curves

In protein quantification, a calibration curve is prepared from the relationship between standard protein concentration and absorbance.
The closer the calibration curve is to a straight line, the easier it is to determine the concentration of the unknown sample.
However, if the concentration is too high, the color reaction may become saturated and the absorbance may deviate from the straight line.

If the absorbance of the unknown sample lies outside the calibration-curve range, the result becomes an extrapolation and the error becomes larger.
In that case, it is desirable to dilute the sample so that it falls within the calibration-curve range.

Causes of Protein Concentration Being Measured as Low

Causes of protein concentration being measured as lower than expected include insufficient extraction, losses during precipitation or centrifugation, protein degradation, dilution errors, errors in the calibration curve, and interference with the color reaction.
Because proteins may denature or degrade because of heat, pH changes, or proteases, sample handling is 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 a negative charge.
Therefore, smaller-molecular-weight proteins move faster through the gel and tend to appear farther down the gel.

In a discussion of SDS-PAGE, explain the position of the target protein band, comparison with the molecular weight marker, band intensity, extra bands, smearing, degree of purification, and presence or absence of degradation.

Discussion When Bands Are Faint

If bands are faint in electrophoresis, possible causes include a small amount of protein or DNA in the sample, insufficient staining, a small sample-loading amount, degradation, or low extraction efficiency.
Bands may also become unclear if the target substance diffuses within the gel or if the electrophoresis conditions are inappropriate.

Discussion When Extra Bands Appear

If extra bands appear in SDS-PAGE or DNA electrophoresis, the sample may contain proteins or DNA fragments other than the target substance.
For proteins, possible causes include incomplete purification, degradation products, and insufficient dissociation of complexes.
For DNA, possible causes include nonspecific PCR products, primer dimers, degraded DNA, and undigested fragments.

Discussion When Smearing Appears

Smearing refers to a condition in which bands are not clearly defined in electrophoresis and instead spread as a blurred signal.
For proteins, possible causes include overloading, degradation, the effects of salt concentration, and poor electrophoresis conditions.
For DNA, possible causes include degradation, overloading, inappropriate gel concentration, excessively high voltage, and impurities in the sample.

Discussion of DNA Extraction Experiments

In DNA extraction experiments, DNA is extracted from cells or tissues, and its concentration, purity, and degree of degradation are evaluated.
DNA extraction involves operations such as cell disruption, protein removal, RNA removal, precipitation, washing, and dissolution.
Losses and contamination with impurities may occur at any stage.

In a discussion of DNA extraction, explain the amount of DNA obtained, absorbance at 260 nm, the 260/280 ratio, electrophoresis bands, presence or absence of smearing, and contamination by proteins or RNA.

Discussion of DNA Concentration and Purity

DNA concentration may be determined from absorbance at 260 nm.
This is because nucleic acids such as DNA and RNA absorb near 260 nm.
Proteins, on the other hand, absorb near 280 nm, so the 260/280 ratio may be used to evaluate DNA purity.

If the 260/280 ratio is low, contamination by proteins or phenol is suspected.
If the 260/280 ratio is too high, RNA contamination or measurement error may be possible.
However, because interpretation of the ratio depends on the measuring instrument and sample conditions, follow the criteria in the laboratory manual.

Causes of Low DNA Yield

Causes of low DNA yield include insufficient cell disruption, insufficient precipitation, loss during supernatant removal, loss during washing, insufficient DNA dissolution, and insufficient sample amount.
In addition, because DNA is a long macromolecule, it may be cut by vigorous stirring or pipetting.

Discussion of PCR Experiments

PCR is a method used to amplify a specific DNA region.
The target DNA fragment is amplified when the DNA template, primers, DNA polymerase, dNTPs, Mg²⁺, buffer, and thermal-cycle conditions are appropriately combined.
After PCR, agarose gel electrophoresis is used to confirm whether a band of the target size is present.

In a discussion of PCR, check for the presence or absence of the target-size band, band intensity, nonspecific bands, primer dimers, and the results of negative and positive controls.

Causes of No Band Appearing in PCR

Causes of failure to obtain the target band in PCR include insufficient template DNA, DNA degradation, problems with primer design, inappropriate annealing temperature, inappropriate Mg²⁺ concentration, enzyme inactivation, inappropriate cycle conditions, and contamination with PCR inhibitors.
In addition, if the electrophoresis or staining conditions are inappropriate, the amplified product may not be visible.

Causes of Nonspecific Bands in PCR

If bands other than the target size appear in PCR, the primers may have bound to sequences other than the target sequence.
If the annealing temperature is too low, primers can more easily bind to partially matching sequences, resulting in nonspecific amplification.
Excessively high primer concentration or Mg²⁺ concentration may also increase nonspecific amplification.

Discussion of DNA Electrophoresis

In DNA electrophoresis, DNA is negatively charged because of its phosphate groups and therefore moves toward the positive electrode in an electric field.
In an agarose gel, smaller DNA fragments pass more easily through the gel network and migrate farther.
By comparing them with a molecular weight marker, the approximate size of the target DNA fragment can be determined.

Effect of Agarose Gel Concentration

Agarose gel concentration affects separation of DNA fragments.
The higher the gel concentration, the finer the gel network becomes, making it suitable for separation of small DNA fragments.
At lower gel concentrations, large DNA fragments migrate more easily, but separation of small fragments may become insufficient.

Common Sources of Error in Protein and DNA Experiments

Protein and DNA experiments have common sources of error.
Representative examples include pipetting errors, sample degradation, inadequate temperature control, reagent deterioration, insufficient mixing, losses during centrifugation, and insufficient calibration of measuring instruments.
Because many biomolecules are sensitive to handling, careful operation greatly affects the results.

Pipetting Errors

In biochemistry experiments, small amounts of samples and reagents are often handled, so errors in pipetting greatly affect the results.
Even slight deviations in sample or reagent volume change enzyme activity, protein concentration, DNA concentration, and PCR reaction conditions.

Particularly with micropipettes, problems include liquid remaining at the tip, aspirating air bubbles, using the wrong plunger position, and difficulty accurately aspirating viscous samples.

Importance of Temperature Control

Temperature control is extremely important in biochemistry experiments.
Enzyme reaction rates change with temperature, and proteins and DNA may denature or degrade when exposed to high temperatures or left for long periods.
If samples that should be stored at low temperature are left at room temperature, activity may decrease or degradation may occur.

Discussion of Blanks and Control Experiments

Blanks and control experiments are important in biochemistry experiments.
Blanks are used to subtract absorbance or color development originating from sources other than the sample.
Negative controls confirm conditions in which no reaction should occur, while positive controls confirm that the reaction system is functioning correctly.

If the control experiments do not give appropriate results, it becomes difficult to draw a correct conclusion from the target sample alone.
In PCR, a band in the negative control suggests contamination, while absence of a band in the positive control may indicate problems with the PCR conditions or reagents.

When the Results Can Be Considered Good

Biochemistry experiment results can be considered good when the standard curve has good linearity, the measured values show reproducibility, the control experiments give the expected results, and the electrophoresis bands match the target size or expected position.
It is also important that enzyme activity, protein concentration, and DNA concentration show theoretically expected trends.

Example Discussion When the Experiment Did Not Go Well

If a biochemistry experiment does not go well, possible causes are considered from results such as a non-linear standard curve, low enzyme activity, absence of a band, extra bands, smearing, or control experiments that differ from expectations.
Rather than ending the discussion with “operational error,” it is easier to discuss the results by separately considering sample condition, reagent conditions, reaction conditions, and measurement methods.

How to Write Points for Improvement

In the discussion of a biochemistry experiment, including points for improvement as well as sources of error makes the report easier to organize.
Improvements are easier to write when divided into sample handling, temperature control, pipetting, measurement conditions, and control experiments.

Improvements to Sample and Temperature Control

• Store samples at an appropriate temperature
• Do not leave enzymes or proteins at room temperature for long periods
• Reduce the number of freeze-thaw cycles
• Perform operations under low-temperature conditions when necessary
• Establish conditions that prevent sample degradation

Improvements to Operations and Measurements

• Use micropipettes accurately
• Prevent contamination by changing tips
• Mix reaction solutions thoroughly
• Standardize reaction times accurately
• Perform blank correction appropriately
• Measure unknown samples within the standard-curve range

Improvements to Electrophoresis and PCR

• Select a gel concentration appropriate for the target size
• Avoid overloading samples
• Use appropriate electrophoresis voltage and time
• Always check positive and negative controls in PCR
• Review primer conditions and annealing temperature
• Prevent degradation of DNA and proteins

Difference Between a Superficial Discussion and a Good Discussion

In the discussion of a biochemistry experiment, simply writing that “enzyme activity was present,” “a band appeared,” or “DNA was extracted” results in a superficial discussion.
It is important to explain what the measured values mean, why the result occurred, and which operations or conditions affected the result.

Examples of Expressions That Can Be Used in Reports

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

• Enzyme activity is affected by temperature, pH, substrate concentration, and enzyme concentration.
• The initial rate was determined from changes in absorbance during the initial stage of the reaction and used to evaluate enzyme activity.
• Under high-temperature conditions, the enzyme protein may have denatured and activity may have decreased.
• Changes in pH altered the charge state of the active site and are considered to have affected substrate binding.
• Because measurement was performed within the linear range of the calibration curve, the concentration conversion is considered generally reasonable.
• Bands other than the target band may originate from impurities or nonspecific reactions.
• Because smearing was observed, the DNA or protein may have been degraded.
• If the 260/280 ratio is low, contamination by proteins or organic solvents may be present.
• Possible causes of failure to obtain the target band in PCR include insufficient template and contamination by inhibitory substances.
• By checking the results of control experiments, the reliability of the target-sample results can be evaluated.

Points to Check When Discussing Biochemistry Experiments

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

• Have you explained what the measured values represent?
• Have you checked the linearity of the standard curve or calibration curve?
• Does the unknown sample fall within the measurement range?
• Have you related enzyme activity to temperature, pH, and substrate concentration?
• Have you discussed protein denaturation and degradation?
• Have you considered DNA purity and the presence or absence of degradation?
• Have you compared electrophoresis bands with a molecular weight marker?
• Have you considered the causes of extra bands and smearing?
• Have you checked blanks and control experiments?
• Have you considered errors in pipetting and dilution?
• Have you considered the effects of sample storage and temperature control?
• Do the points for improvement correspond to the sources of error?

Summary

In biochemistry laboratory reports, because biomolecules such as enzymes, proteins, and DNA are handled, it is important to discuss not only measured values but also sample conditions and reaction conditions.
In enzyme experiments, temperature, pH, substrate concentration, and enzyme concentration affect activity.
In protein experiments, consider calibration curves, color reactions, denaturation, degradation, and the effects of impurities.
In DNA experiments, check extraction efficiency, purity, degradation, PCR conditions, and electrophoresis bands.

In biochemistry experiments, pipetting, temperature control, sample storage, blank correction, and control experiments determine the reliability of the results.
In particular, biomolecules are susceptible to denaturation and degradation, so the conditions during operations are strongly reflected in the results.

In a report, do not simply write that “enzyme activity was present,” “protein was detected,” or “a DNA band appeared.”
Explain why the result occurred in relation to the reaction conditions, sample condition, measurement method, and sources of error.
Relating the results to the operating conditions produces a more persuasive biochemistry laboratory report.