One of the most important techniques in molecular biology is Western blotting. Used to identify and separate proteins extracted from cells for study, the method has existed for decades, refined from earlier blotting methods to become one of the most common in science. However, Western blotting has some serious drawbacks in its application, and nowhere is this truer than in the reproducibility of data.
Reproducibility Problems in Scientific Data
A critical aspect of any good study is how reproducible it is. Can other scientists mimic the steps taken to do their own studies? Can they confirm findings? Can they use the techniques showcased in the study to craft medical procedures?
Unfortunately, failure to reproduce findings is not uncommon. That, of course, makes life difficult for researchers. When the answer is no – as it is a significant percentage of the time – problems arise with credibility, not to mention the simple frustration of data unconfirmed by outside sources.
The good news is that this isn’t an irrevocable condition. While Western blotting does have issues that make reproducing data difficult, there are steps that can be taken to reduce them.
Take the Utmost Precaution with Lysis Methods
Cell lysis (the breaking open of cells in order to extract their biological contents) is fundamental to Western blotting. Without careful lysis methods, the cell’s innards can get damaged, degraded or contaminated. Any of these will make the sample unusable or potentially difficult to reproduce. To prevent this, researchers should use:
- Well-vetted lysis buffers that enable the breakdown of cell membrane without damaging the organelles within
- Cold materials that minimize damage to cell contents
- Clean and pH-neutral tools and water
- Ready access to flash-freezing and storage
Other concerns when it comes to optimizing lysis methods include what types of proteins you’re trying to extract; where they are located; and whether the methods will cause foaming, which can damage proteins.
Select the Correct Gel Type for Electrophoresis
Electrophoresis is a relatively simple method for separating out proteins. By using a specific type of gel with pores, and running a current through it, the molecules will stratify by electrical charge. Smaller molecules can move farther through the pores than larger molecules, leaving the various components of the cell in distinct groups. This makes it easy for researchers to isolate the target protein.
Much like lysis methods, the gel type has a significant bearing on the quality of the sample. You will compound the success of the former by using the correct gel during the next step of the process.
As with lysis methods, gel types require knowing the size of your target proteins, whether the size difference between target and non-target proteins is broad or narrow, how many samples you want to run, and more.
Choose the Correct Blotting and Transfer Materials/Techniques
The transfer step is another point at which problems can arise. Separated proteins must be moved from the gel to a membrane for study, but this can cause breakage or inhibit binding if not done correctly.
It’s critical to match the blotting and transfer materials to the goals of the study. That means choosing between wet, semi-dry and rapid semi-dry methods. Each offers different benefits in sample preservation and speed.
Optimize Antibody Methods for Reproducibility
Antibodies are critical to Western blotting success. Primary antibodies bind to the proteins once they’ve been transferred to the membrane, which is then washed to remove excess antibodies. Secondary antibodies, which have been treated to make protein identification possible, are then introduced as well, binding to the primary antibody.
In order to do this, antibodies must have a strong signal to the target protein and are specific only to it, so that they don’t bind to other undesirable proteins. Running tests beforehand to ensure this is critical, otherwise, the main experiment may be contaminated.
Collect and Verify Data with Best Practices
Measurement is, in the end, the entire point of any experiment. After the sample is prepared completely, you will use software to measure protein bands produced by Western blots. Image analysis can remove backgrounds and clarify the samples, enabling scientists to compare samples to one another.
Normalization is an important aspect of this, balancing out minor deviations in the preparation, gel loading and transfer of the sample. This rules out normal variability in the data and ensures that any results recorded are that of the experimental conditions, both validating data and making its reproduction easier later on.
Western blotting is a complex process that takes several days, but you can make those days worth it with the creation of valuable, reproducible data, so take these steps today.