Step-by-Step Guide to Quantifying Western Blot Protein Bands: Tools and Techniques

The technique of western blotting involves several key steps. Briefly, it involves sample preparation, gel electrophoresis of the isolated proteins from the samples, followed by transfer of the proteins onto a more durable membrane (nitrocellulose or PVDF).  The membrane is then blocked to avoid nonspecific interactions, in the subsequent step of adding the target protein antibody. 

Following incubation of the membrane-bound protein with the target antibody, the presence and quantity of the protein (s) is verified. 

For a detailed walkthrough of how to quantify the detected protein on the nitrocellulose or PVDF membrane, see my YouTube video embedded below. 

Quantifying western blot bands using image analysis software, is an essential step to determine the relative expression levels of proteins. There are several software that may be used. Common options include Image J (Fiji), GelAnalyzer and proprietary software from imaging systems such as BioRad's Image Lab. Below are the expanded steps to perform accurate quantification in ImageJ.

Step 1
Obtain a high-quality image: use a digital imaging system or scanner to capture the blot. Ensure the image is well-exposed and not over-saturated. 
Step 1.1
Format and Resolution: save the image in a high-resolution format (e.g., TIFF) for better accuracy during 

Step 1.2
Prepare the Image for Analysis: Transform the gel image if you find that it's upside down or it's not in the right orientation after opening the file. To do this, go to Transform and rotate to flip it right around. 
Step 1.3
Convert the image to Grayscale: convert the image to grayscale, as most analysis tools work on intensity values in black-and-white images. 
Step 1.4
Adjust the Brightness and Contrast: optimize brightness and contrast for clear visualization of bands without introducing artifacts. 
Step 1.5
Crop the Image: crop unnecessary parts of the image to focus on the lanes and bands being analyzed, avoiding marker lanes if not relevant. 
Step 1.6
There is an option to invert the image. This allows the peaks that you are about to detect and quantify, to appear upside down, so that you can draw a line at the base of the peak for quantitation. 

Define the Region of Interest (ROI) 

Step 2

Mark Bands: Draw rectangular or elliptical ROIs around each band you want to quantify. In ImageJ, I use the same rectangle to quantify each of the bands. To enable this, I first move the rectangle to all the bands and adjust it to ensure that it's the right width and height to capture each band. 

Step 2.1 
Measure band intensity In ImageJ, you do so by selecting --> Analyze. In Analyze you go right down to the specific option which says ---> Gels and then you can select the first lane. After selecting the first lane, use the forward arrow on your keypad and it will duplicate that rectangle. Move to the next band/lane with the duplicated rectangle, then select ---> Analyze ---> Gels. This time, you must choose --> 'select next lane' rather than 'select first lane'. Repeat with --> 'select next lane' while using the arrows on your keyboard to move to all the bands that you want to quantify. Once you've quantified all the bands using the duplicated rectangular tool, you want to Plot Lanes. 

Step 2.2
Go back to --> Analyze --> Gels and then this time, choose ---> Plot Lanes. The software will now plot the peaks to match the intensities it has detected in each lane. Once you have your peaks you want to draw the boundary of each peak. This is why it is useful to have inverted the image. With the inverted image, the peak will appear upside down, allowing you to see the boundaries of the peak clearly. 
Draw a line to demarcate where a peak begins and ends. Once you have the peak clearly marked, you select the wand tool. 
Using the wand tool, click on the peaks that you have drawn, to obtain the pixel intensity within each band. 

Correct for Background: measure an empty region near any of the bands to use for background correction. Copy the numerical output of each peak (and the background peak) and paste the outputs in Excel or your data analysis tool of choice.
Subtract the background intensity value from each of the peak intensities. 

Normalise data using the loading control: normalise band intensities to a housekeeping protein such as beta-actin or GAPDH to correct for variations in loading and transfer. 

Relative Quantification: divide the band intensity of the target protein by the intensity of the loading control in the same lane. 

Plot and Analyze Results 

Step 3

Create Graphs: plot normalised band intensities as bar graphs or scatter plots using software such as Excel or Graphpad Prism.

Step 3.1
Statistical Analysis: perform statistical tests such as t-tests or ANOVA to determine the significance of differences between groups.

LOG2 FROM LOG10 ON A CALCULATOR

Two years ago, at the request of a subscriber, I posted a tutorial on how to perform log2 transformations on a calculator. This request stems from the fact that you typically have your default  button for logarithmic transformations, assuming that you want a log10 transformation. Even in Excel, the default log function, assumes that you want a log10. When this is not the case, how do you get a log2  transformation?

For a detailed walkthrough of this process, check out my YouTube video embedded below.

Why might you want a log2 transformation, rather than the default log10?

Advantages of Log2 Transformation

1. Interpretability in Fold-Changes:

- Log2 transformation is particularly useful in biological and genomic studies, where changes in gene expression, protein levels, or other measurements are often discussed in terms of fold-changes. A log2 transformation makes the interpretation straightforward: - A value of 1 corresponds to a 2-fold increase. - A value of -1 corresponds to a 2-fold decrease. - A value of 0 indicates no change. - This symmetry around zero (up-regulation and down-regulation) is intuitive and simplifies analysis.

2. Symmetry for Data Centered Around Ratios:

- In datasets where the focus is on ratios (e.g., treatment vs. control), log2 transformation ensures that up-regulations and down-regulations are equally scaled, facilitating statistical analysis and visualization.

3. Compatibility with Software and Models:

- Many computational pipelines and bioinformatics tools are designed to handle log2-transformed data, making it the standard for certain fields.

4. Reduced Skewness:

- Like other log transformations, log2 can help reduce skewness in data, bringing distributions closer to normality and stabilizing variances across a range of values. Why Not Log10 or Natural Log (ln)? - Log10 (log base 10) is more common in engineering or chemical studies, where orders of magnitude are the focus. However, it is less intuitive for interpreting fold changes. - The natural logarithm (ln, log base e) is useful in mathematical modeling and continuous systems but lacks the fold-change interpretation directly tied to binary doubling/halving. Log2 transformation is often the most practical choice in biological studies where fold changes and symmetry around a reference point (like "no change") are critical for analysis and visualization. It is more intuitive and directly applicable in these contexts compared to log10 or ln.

Step-by-Step Guide: Culturing Plasmodium falciparum Strains in the Lab

 

Culturing Plasmodium falciparum Strains in the Lab 

Culturing Plasmodium falciparum in the lab is a critical process for malaria research. This article provides the instructions for culturing laboratory strains of Plasmodium falciparum, the deadliest species of malaria parasite.
The cells are typically cultured in vitro using human erythrocytes (red blood cells, RBCs). The culture system requires precise control of environmental conditions, including temperature, gas composition, and nutrient availability.
For a detailed walkthrough of this protocol, check out my YouTube video embedded below!  

Click to watch my video on culturing P. falciparum strains!

Disclaimer 

Culturing Plasmodium falciparum is a highly specialized technique that requires extensive training, biosafety level 2 or 3 containment, and access to specialised equipment.
Culturing Plasmodium parasites, including Plasmodium falciparum, requires strict adherence to safety precautions due to the risks associated with handling pathogens and human blood products. Key safety measures include:

1. Biosafety Level 2 (BSL-2) Facilities: Labs must operate under BSL-2 conditions when working with Plasmodium cultures. This includes the use of biological safety cabinets for procedures that generate aerosols, proper ventilation, and restricted access to the laboratory to limit exposure.
2. Personal Protective Equipment (PPE):  Laboratory personnel should wear gloves, lab coats and eye protection. additional protective measures, such as face shield, may be necessary when handling cryopreserved samples or during procedures involving liquid nitrogen.
3. Handling of Human Blood: Since human red blood cells are used in the culture medium, there is a risk of transmitting bloodborne pathogens. All blood samples should be treated as potentially infectious, sourced from accredited suppliers, and screened for pathogens like HIV and hepatitis.
4. Decontamination Procedures: Spills, waste, and contaminated materials should be decontaminated using appropriate disinfectants, such as 10% bleach or autoclaving. Waste disposal must follow regulatory guidelines for biohazardous materials.
5. Cryopreservation Risks: When thawing cryopreserved Plasmodium samples, handle under sterile conditions to prevent cross-contamination.
6. Aerosol Generation Control: Manipulations that could generate aerosols should be minimised, as they pose a risk of infection. Procedures such as centrifugation must use sealed rotors or safety cups to contain aerosols.
7. Training and Documentation: Personnel must be trained in handling infectious agents and in emergency protocols. Laboratory procedures should be well-documented, and safety data sheets for all chemicals used must be readily accessible.