Interview Questions for Immunoassays (ELISA, Western Blotting)

ELISA, or Enzyme-Linked Immunosorbent Assay, is a powerful technique used to detect and quantify the presence of a specific substance, typically a protein or antibody, in a liquid sample. It works on the principle of antigen-antibody binding. Imagine a lock and key: the antigen (the substance you’re looking for) is the lock, and the antibody (a highly specific protein that binds to the antigen) is the key. The assay uses an enzyme linked to an antibody, which, upon binding to the antigen, catalyzes a colorimetric or fluorometric reaction, enabling detection and quantification.

The intensity of the color or fluorescence is directly proportional to the amount of antigen present in the sample. This allows us to determine not only if the target substance is present but also how much of it is there.

There are several types of ELISA, each with its own advantages and disadvantages:

• Direct ELISA: This is the simplest type. An antibody directly conjugated to an enzyme is added to the sample. If the antigen is present, the antibody binds, and upon addition of the substrate, a detectable signal is generated. Think of it as directly labeling the ‘lock’ with a color-changing ‘key’.
• Indirect ELISA: This method uses a primary antibody to bind the antigen, followed by a secondary antibody (conjugated to an enzyme) that binds to the primary antibody. This amplifies the signal, making it more sensitive. This is like using a ‘helper key’ to detect the first key’s binding.
• Sandwich ELISA: This is particularly useful for detecting antigens. A capture antibody is immobilized on the plate, followed by the addition of the sample. If the antigen is present, it binds to the capture antibody. Then, a detection antibody (conjugated to an enzyme) binds to a different epitope on the antigen, creating a ‘sandwich’ structure. This method is highly specific and sensitive. It’s like trapping the ‘lock’ between two specific ‘keys’.
• Competitive ELISA: This method is used to measure the concentration of an antigen in a sample by competing with a known concentration of labeled antigen for binding to a limited number of antibodies. The less signal produced, the higher the concentration of antigen in the sample. This is like having two ‘keys’ competing to fit into the same ‘lock’.

The steps involved in a typical ELISA (taking the indirect ELISA as an example) are:

1. Coating: The microplate wells are coated with the antigen.
2. Blocking: A blocking agent (e.g., BSA) is added to prevent non-specific binding of antibodies to the plate.
3. Sample addition: The sample containing the potential antibody is added to the wells.
4. Primary antibody incubation: The primary antibody binds to the antigen if present.
5. Washing: Unbound antibodies are removed by washing.
6. Secondary antibody incubation: The enzyme-conjugated secondary antibody binds to the primary antibody.
7. Washing: Unbound secondary antibody is removed by washing.
8. Substrate addition: The substrate is added, leading to a color change proportional to the amount of bound antibody.
9. Measurement: The absorbance is measured using a plate reader.

Accuracy and reproducibility in ELISA are paramount. Several measures ensure this:

• Using high-quality reagents: Using validated reagents from reputable suppliers is crucial.
• Proper pipetting techniques: Precise and consistent pipetting avoids variations in reagent volumes.
• Appropriate controls: Including positive and negative controls, as well as blank wells, helps validate the assay’s performance and correct for background noise.
• Standard curve: A standard curve using known concentrations of the analyte is essential for accurate quantification.
• Using a validated protocol: Following established and validated protocols is essential for reproducibility.
• Maintaining consistent temperature and incubation times: Temperature and time affect antibody binding and enzyme activity.
• Precise washing steps: Efficient washing steps remove unbound reagents to avoid interference.
• Proper data analysis: Correct data analysis using appropriate software and statistical methods is crucial.

Common sources of error in ELISA and their minimization:

• Non-specific binding: Minimized by using effective blocking agents and careful washing.
• Improper washing: Insufficient washing leads to high background noise. Ensure complete removal of unbound reagents using appropriate wash buffers and procedures.
• Incorrect pipetting: Leading to inconsistent reagent volumes; practice proper pipetting techniques and use calibrated pipettes.
• Degradation of reagents: Using fresh reagents and following storage instructions prevents reagent degradation.
• Plate reader variations: Calibrating the plate reader regularly ensures consistent readings; using the same plate reader across experiments enhances reproducibility.
• Temperature fluctuations: Maintaining constant temperatures during incubations prevents variation; use incubators to maintain a stable temperature.
• Human error: Following a detailed protocol and using quality control checks helps to avoid human error.

Western blotting is a powerful technique used to detect specific proteins within a complex mixture of proteins, such as a cell lysate. The principle relies on separating proteins by size using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis), transferring the separated proteins onto a membrane, and then using antibodies to detect the protein of interest. Think of it as a ‘protein fingerprint’ identification method. SDS-PAGE separates the proteins based on their molecular weight, creating bands. The antibodies then specifically bind to their target protein, allowing for identification and quantification.

The steps involved in a Western blot are:

1. Sample preparation: Cell or tissue lysates are prepared by lysis and centrifugation.
2. SDS-PAGE: Proteins are separated by size using SDS-PAGE.
3. Transfer: Proteins are transferred from the gel to a membrane (typically nitrocellulose or PVDF).
4. Blocking: The membrane is blocked to prevent non-specific antibody binding.
5. Incubation with primary antibody: The membrane is incubated with a primary antibody specific to the target protein.
6. Washing: Unbound primary antibody is washed away.
7. Incubation with secondary antibody: The membrane is incubated with a secondary antibody conjugated to an enzyme (e.g., horseradish peroxidase) or a fluorescent label, which binds to the primary antibody.
8. Washing: Excess secondary antibody is washed away.
9. Detection: The target protein is detected using chemiluminescence, fluorescence, or colorimetric methods. The resulting bands indicate the presence and size of the target protein.

Western blotting, also known as immunoblotting, is a powerful technique used to detect specific proteins in a complex sample. While the most common type involves transferring proteins from a gel to a membrane, several blotting techniques exist, each suited to different experimental needs.

• Electrophoretic transfer: This is the most prevalent method. Proteins separated by SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis) are transferred onto a membrane (typically nitrocellulose or PVDF) using an electric field. This transfer process maintains the separation achieved during electrophoresis, allowing for identification of specific proteins based on their size.
• Capillary blotting: This is a simpler, less efficient method involving passive transfer of proteins from the gel to the membrane via capillary action. It’s less common now due to its slower speed and less even transfer compared to electrophoretic blotting.
• Vacuum blotting: Vacuum blotting accelerates the transfer process compared to traditional electrophoretic transfer by using a vacuum to draw the buffer and proteins through the gel and onto the membrane. This can lead to more efficient transfer and improved signal strength.
• Semi-dry blotting: This method uses lower voltage and less buffer volume than traditional wet blotting. It’s quicker and uses less equipment, making it a convenient choice for many labs.

The choice of blotting technique depends on factors such as the size of the proteins, the type of membrane used, and the available equipment. Electrophoretic blotting is generally preferred for its efficiency and reproducibility.

Interpreting Western blot results requires careful attention to detail. The primary goal is to identify the presence and size of your target protein.

• Bands: The presence of a band at the expected molecular weight (MW) indicates the presence of your target protein. The intensity of the band reflects the amount of the protein present in the sample.
• Molecular Weight: Compare the band’s position to a protein ladder (molecular weight marker) to estimate the protein’s molecular weight. Discrepancies between expected and observed MW can indicate post-translational modifications or protein degradation.
• Positive and Negative Controls: Positive controls (samples known to contain the target protein) should show bands at the expected MW, validating the assay. Negative controls (samples known to lack the target protein) should not show bands, indicating specificity of your antibodies.
• Non-specific binding: Observe for non-specific bands at other positions. These may be due to non-specific antibody binding or other issues like contamination.

Quantifying band intensity (using densitometry) can provide semi-quantitative data about protein expression levels. It’s important to remember that Western blotting is not highly quantitative, and results should be carefully interpreted in the context of the experimental design and controls.

For instance, a strong band in a treated sample compared to a weak band in a control sample indicates increased protein expression in the treated condition. Absence of a band in the sample where you expected to see your protein indicates the protein is either absent or present in very low levels, possibly below the detection limit of the assay. You might need to optimize the assay or use a more sensitive detection method.

Western blotting is a complex technique prone to troubleshooting. Here’s a systematic approach:

• No bands visible (or very faint bands): Check antibody concentration, incubation times, blocking efficiency, substrate sensitivity and detection method. Ensure the protein was successfully transferred, that your antibodies are specific and working properly, and that there wasn’t significant protein degradation.
• Non-specific bands: Try increasing the blocking concentration or time, using a different blocking agent, or using higher affinity antibodies. Consider using secondary antibodies with lower cross-reactivity.
• Smearing or uneven transfer: This may be due to poor transfer conditions (voltage, time, buffer composition), incorrect gel composition, or protein degradation. Re-evaluate your transfer parameters and ensure the gel isn’t damaged.
• High background: Check for contamination, non-specific antibody binding, insufficient washing, or inadequate blocking. Ensure all reagents are fresh and stored properly.
• Incorrect molecular weight: Verify the predicted protein size, and consider potential post-translational modifications that might alter the size of your protein. Check the quality of the protein ladder.

Troubleshooting effectively requires a methodical investigation, starting from the basic parameters and progressing systematically. Keeping detailed records throughout the experiment is essential for effective troubleshooting.

ELISA (Enzyme-Linked Immunosorbent Assay) is a versatile technique used widely in research and diagnostics for detecting and quantifying substances such as proteins, peptides, antibodies, and hormones.

• Research applications include measuring cytokine production in cell culture, studying antibody responses to vaccines, and analyzing protein-protein interactions. For example, researchers can use ELISA to quantify the amount of a specific cytokine released by immune cells stimulated with a particular antigen.
• Diagnostic applications are numerous. ELISA tests are crucial for diagnosing infectious diseases such as HIV and Hepatitis B and C, detecting pregnancy (human chorionic gonadotropin, hCG), and identifying autoimmune diseases. Home pregnancy tests are a common example of a direct ELISA-based diagnostic.

The ability to automate ELISA makes it a high-throughput method, suitable for large-scale screening and diagnostics.

Western blotting is a technique with significant applications in both research and diagnostics.

• Research applications: It’s essential for studying protein expression levels, post-translational modifications, and protein-protein interactions. For instance, Western blotting can be used to detect changes in protein expression levels in response to a drug treatment, and identify the proteins involved in a signaling pathway.
• Diagnostic applications: While less frequently used than ELISA for routine diagnostics due to its higher complexity, it’s important for confirming the presence of specific proteins, especially in cases where high specificity is required. For example, it can be used to detect the presence of specific disease markers, but typically in a more specialized lab setting compared to the broader utility of ELISA.

The ability of Western blotting to determine the size and quantity of a specific protein makes it a powerful tool for evaluating protein expression patterns.

Selecting appropriate antibodies is crucial for successful ELISA and Western blotting. The key considerations are specificity, affinity, and isotype.

• Specificity: The antibody must bind exclusively to the target protein or antigen, minimizing cross-reactivity with other proteins in the sample. Data sheets and validation from the supplier are vital.
• Affinity: High-affinity antibodies bind tightly to the target, resulting in stronger signals and improved sensitivity.
• Isotype: The antibody isotype (e.g., IgG, IgM) determines which secondary antibody is needed and influences the signal strength.
• Antibody validation: Look for published studies validating the antibody’s performance in ELISA or Western blotting, noting whether applications are for ELISA or Western blotting, as some antibodies may work well in one assay but not the other.
• Antibody source: Check for the source (monoclonal vs. polyclonal), as monoclonal antibodies typically offer higher specificity but may be more expensive. Polyclonal antibodies can be less expensive but may exhibit greater cross-reactivity.

Careful selection of antibodies significantly impacts the reproducibility and reliability of your results. Always check the antibody’s specifications before making a purchase.

Optimizing antibody and antigen concentrations is critical for achieving optimal signal-to-noise ratios in ELISA and Western blotting. This usually involves performing titration experiments.

• Antibody titration: Perform serial dilutions of the primary and secondary antibodies to determine the optimal concentrations that yield strong signals with minimal background noise. This is often represented as a checkerboard titration for ELISA. The goal is to find the “sweet spot” – the concentration that offers the best signal while minimizing non-specific binding.
• Antigen titration (ELISA): For ELISA, you may need to optimize the concentration of the antigen coated on the plate to ensure adequate binding without saturation. Too little antigen might produce weak signals, while too much can lead to high background.
• Sample preparation and loading (Western blotting): For Western blotting, protein sample concentration should be optimized to avoid overloaded lanes where the bands may not be well-resolved.
• Visual and quantitative analysis: Evaluate the results visually (e.g., band intensity in Western blot or color development in ELISA) and potentially use quantitative analysis (e.g., densitometry) to precisely determine the optimal concentrations.

Systematic optimization ensures the assay is working at its best, giving you reliable and reproducible results. A well-optimized assay will produce consistent results, minimizing variations between experiments and improving data interpretation.

Standardization and calibration in ELISA are crucial for obtaining reliable and comparable results. Think of it like baking a cake – you need a consistent recipe (standardization) and a way to measure the ingredients accurately (calibration) to ensure the cake comes out the same every time. In ELISA, standardization involves using a standard curve, which is a graph plotting known concentrations of the analyte (the substance you’re measuring) against their corresponding absorbance values. This curve is generated using a set of standards with known concentrations. Calibration, on the other hand, refers to the process of adjusting the instrument (e.g., a plate reader) to accurately read the absorbance values. It ensures that the instrument’s readings are consistent and accurate. Without proper standardization and calibration, your ELISA results would be meaningless, as you wouldn’t be able to accurately determine the concentration of the analyte in your samples.

For example, if you’re measuring the concentration of a specific hormone in blood samples, you’ll need a standard curve generated using known concentrations of that hormone. Each sample’s absorbance is then compared to this curve to determine its concentration. Regular calibration of your plate reader is vital to ensure the absorbance readings are accurate and consistent throughout the experiment.

ELISA employs various detection methods, each with its own strengths and weaknesses. The choice depends on factors like sensitivity and cost. Common detection methods include:

• Colorimetric: This is the most common method, relying on a color change proportional to the amount of analyte. A common substrate is TMB (3,3′,5,5′-Tetramethylbenzidine), which produces a blue color that turns yellow upon acidification. The absorbance is then measured using a plate reader. It’s relatively inexpensive and easy to perform.
• Fluorometric: This method uses fluorescent substrates, which emit light at a specific wavelength when excited by another wavelength. It offers higher sensitivity than colorimetric methods because fluorescence can be measured with less background noise. Examples include fluorescein isothiocyanate (FITC) and R-phycoerythrin (R-PE).
• Chemiluminescent: This method involves substrates that produce light upon a chemical reaction. It’s known for its high sensitivity and dynamic range. Examples include luminol and acridinium esters.
• Enzymatic: This method uses enzymes as labels, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). These enzymes catalyze a reaction that produces a detectable signal (colorimetric, fluorometric, or chemiluminescent).

Imagine choosing the right tool for a job. For a routine assay needing a quick and cost-effective method, colorimetric might be ideal. If higher sensitivity is a priority, fluorometric or chemiluminescent methods would be preferred.

Western blotting detection methods primarily focus on visualizing the protein bands after transfer to a membrane. The most common detection methods are:

• Chemiluminescence: This is a widely used method employing HRP or AP conjugated secondary antibodies. The enzyme catalyzes a reaction producing light, which is detected by X-ray film or a chemiluminescence imager. It’s sensitive and provides a good dynamic range.
• Colorimetric: Similar to ELISA, colorimetric detection uses substrates that yield a colored product upon enzymatic activity. This approach is less sensitive than chemiluminescence but simpler and can be visually assessed.
• Fluorescent: Fluorescently labeled secondary antibodies offer high sensitivity and the ability to detect multiple proteins simultaneously (multiplexing). Specialized imaging systems are needed for detection.
• Radioactive: Although less common now due to safety concerns and disposal, radioactive isotopes can be used to label proteins. Autoradiography then reveals the protein bands.

The choice depends on factors such as sensitivity requirements, availability of equipment, and safety concerns. For example, if high sensitivity is crucial and cost isn’t a major constraint, chemiluminescence or fluorescence would be the preferred choices.

Calculating analyte concentration from ELISA data involves creating a standard curve and using it to interpolate the concentrations of unknown samples. First, plot the absorbance values of your standards (known concentrations) against their respective concentrations on a graph (typically a logarithmic scale for the x-axis). This creates a standard curve, usually a sigmoid curve. Then, measure the absorbance of your unknown samples. Find the absorbance values on the y-axis of your standard curve and determine the corresponding concentration on the x-axis. This interpolated value represents the approximate concentration of the analyte in your samples.

For example, if your sample’s absorbance is 0.8, and your standard curve shows that an absorbance of 0.8 corresponds to a concentration of 5 ng/mL, then the estimated concentration of your sample is 5 ng/mL. Most ELISA plate readers have software to automate this calculation. It’s crucial to use appropriate statistical methods to account for experimental variability and ensure reliable results.

The intensity of bands in a Western blot reflects the amount of the target protein present in the sample. A darker band indicates a higher protein concentration, while a lighter band or absence of a band suggests a lower or absent protein concentration, respectively. Qualitative assessment is visual – comparing the band intensity across different lanes (samples) provides relative comparisons. However, accurate quantification necessitates densitometric analysis.

For instance, if you are comparing protein expression levels in control and treated cells, a significantly darker band in the treated cells compared to the control cells suggests increased protein expression. It’s crucial to remember that equal protein loading is essential for fair comparisons. Loading controls (like beta-actin) help verify this.

Quantifying protein expression using Western blotting usually involves densitometry, a technique that measures the intensity of bands on the blot. Software analyzes the digital image of the blot, measuring the pixel density within each band. This density is directly proportional to the amount of protein present. The intensity of the target protein band is then normalized to a loading control (e.g., housekeeping protein like GAPDH or β-actin) to account for differences in sample loading. The ratio of target protein intensity to loading control intensity provides a quantitative measure of protein expression.

Imagine you have two bands – one representing your protein of interest and another representing a loading control. Densitometry software measures the pixel intensity of both. The ratio of the intensity of the protein of interest to the loading control corrects for any variations in sample loading and provides a more accurate estimate of protein expression. This normalized data is then statistically analyzed to determine significant changes in protein expression.

Both ELISA and Western blotting are powerful techniques for analyzing biomolecules, but they have distinct advantages and disadvantages:

• ELISA:
• Advantages: High-throughput, relatively quick, simple to perform, quantitative, cost-effective for large sample numbers.
• Disadvantages: Can be less sensitive than Western blotting, requires purified antigen, may suffer from cross-reactivity.
• Western Blotting:
• Advantages: High sensitivity, can detect multiple proteins simultaneously (with modifications), provides information about protein size and post-translational modifications.
• Disadvantages: Lower throughput, more time-consuming, technically demanding, less quantitative than ELISA, requires more specialized equipment.

Think of it as choosing the right tool for the job. If you need to quickly screen a large number of samples for a specific protein, ELISA might be the best option. However, if you need high sensitivity and information about protein size and modifications, Western blotting is generally preferred.

ELISA (Enzyme-Linked Immunosorbent Assay) and Western blotting are both powerful techniques used to detect specific proteins, but they differ significantly in their approach and application. ELISA is a plate-based assay that measures the presence of a specific protein in a liquid sample (e.g., serum, cell lysate) by using antibodies. It’s relatively quick, high-throughput, and quantitatively measures the amount of protein present. Western blotting, on the other hand, is a more complex technique that separates proteins by size using gel electrophoresis before transferring them to a membrane. Antibodies are then used to detect the target protein, allowing for visualization and confirmation of protein size and presence. This makes it more qualitative but useful for confirming protein identity and modification.

• ELISA: Think of it like a highly sensitive color-changing thermometer. If your target protein is present, it binds to the antibody, triggering a color change proportional to the amount of protein.
• Western blotting: Imagine sorting candies by size before picking out your favorite one. The gel separates proteins by size, the membrane acts as the candy display, and the antibody helps you pinpoint your specific target protein.

In summary, ELISA excels in high-throughput quantitative analysis of a protein in a sample, while Western blotting is better suited for qualitative confirmation of protein identity and molecular weight, as well as identifying protein modifications like phosphorylation.

Data analysis in ELISA and Western blotting often involves specialized software. For ELISA, I commonly use plate reader software provided by the manufacturer to obtain raw absorbance values. These data are then exported to software packages like GraphPad Prism or Excel for further analysis, including calculating means, standard deviations, and performing statistical tests like t-tests or ANOVA. For Western blotting, image analysis software such as ImageJ or Image Lab is crucial. These programs allow for quantification of band intensity after normalization to a loading control, enabling comparisons between samples. Sometimes, more advanced software may be used for automated data processing and image analysis.

The choice of software depends largely on the specific needs of the experiment and the available resources.

Troubleshooting in immunoassays requires a systematic approach. In ELISA, common issues include high background, low signal, or inconsistent results. I’ve encountered high background due to non-specific binding which I addressed by optimizing blocking conditions, using lower antibody concentrations or switching to a different blocking agent. Low signals might indicate problems with antibody affinity, concentration, or incubation times, necessitating optimization of these parameters. Inconsistent results are usually addressed by checking for variability in pipetting, plate washing efficiency, and ensuring correct temperature control throughout the assay.

For Western blotting, troubleshooting commonly focuses on issues such as poor transfer, non-specific binding, or faint or absent bands. Poor transfer can be addressed by verifying proper transfer conditions (voltage, time), while non-specific binding can often be minimized by using blocking solutions, optimizing antibody concentrations, or employing other blocking techniques. Faint bands indicate a need for concentration optimization or antibody affinity enhancement. In my experience, meticulously documenting each step and carefully considering each potential source of error is crucial for effective troubleshooting.

Safety and compliance are paramount in my work. I strictly adhere to all relevant laboratory safety regulations, including the proper handling and disposal of biological materials, the use of personal protective equipment (PPE), and working under a biosafety hood when necessary. All reagents used are handled according to the manufacturer’s safety data sheets (SDS). For example, I always work with hazardous materials such as certain antibodies or blocking agents inside a biosafety cabinet, properly labeled and with appropriate waste disposal protocols followed. Furthermore, I maintain meticulous records of all experiments, including reagent details, protocols, and results to ensure traceability and reproducibility, adhering to good laboratory practices (GLP) and relevant regulatory standards like those in place with my specific research institution.

I have experience with various ELISA plate readers, including absorbance readers (measuring optical density at specific wavelengths), fluorescence readers (detecting fluorescence emission), and luminescence readers (measuring light emission). Absorbance readers are most commonly used in standard ELISA using colorimetric substrates. Fluorescence readers offer increased sensitivity and are used in fluorescence-based assays. Luminescence readers provide exceptional sensitivity and are used in chemiluminescent or bioluminescent ELISA. Each reader has specific functionalities, such as wavelength selection, shaking capabilities, and data output options which allow flexibility to choose the appropriate one based on assay type and desired sensitivity.

For example, I have extensively utilized a BioTek Synergy HTX multi-mode plate reader capable of all three detection modalities, allowing adaptation to many experimental setups. The software associated with these readers plays a crucial role in data acquisition and analysis.

Optimizing an ELISA for increased sensitivity and specificity involves a multi-faceted approach. To enhance sensitivity, you can explore several strategies: 1) Optimization of antibody concentrations: Finding the optimal concentration of both capture and detection antibodies through titration experiments is crucial. 2) Signal amplification strategies: Employing techniques such as biotin-streptavidin amplification or enzyme-linked secondary antibody systems can significantly enhance the signal. 3) Substrate selection: Selecting a substrate with high sensitivity and low background is key.

To improve specificity, you need to minimize non-specific binding. 1) Optimization of blocking conditions: Thorough blocking of the plate surface using suitable blocking agents like BSA or casein reduces non-specific binding. 2) Antibody optimization: Selecting antibodies with high affinity and specificity for the target antigen is critical; antibodies from different clones may be evaluated to compare their performance. 3) Washing steps: Careful optimization of washing steps removes unbound material, reducing background noise. By systematically optimizing these parameters, we can achieve significant improvements in assay performance.

Assay validation is a critical step to ensure reliability and reproducibility. For ELISA, validation involves assessing parameters such as linearity, sensitivity (limit of detection and quantification), accuracy, precision (intra- and inter-assay variability), and specificity (cross-reactivity). This usually involves running the assay with several different concentrations of the target analyte and appropriate controls. For Western blotting, validation focuses on demonstrating the specificity of the antibody by ensuring it binds only to the target protein. This involves using positive and negative controls, appropriate loading controls, and potentially evaluating the assay’s ability to detect different protein isoforms or modifications. In both assays, generating robust validation data according to established guidelines provides confidence in the assay’s performance and allows its reliable application in subsequent experiments. Careful documentation of all validation experiments, including detailed protocols and raw data is essential for good laboratory practice and ensures the reproducibility of the assay.