Western Blotting Normalization Required to Achieve Publishable Results
When W. Neal Burnette submitted his method for protein analysis to Analytical Biochemistry in 1979, reviewers rejected it, labeling the technique as “pedestrian.” But word-of-mouth notoriety took over, and the western blotting technique gained popularity among biologists.
“The few preprints I had sent to colleagues seemed to have undergone logarithmic Xerox multiplication. I began receiving phone calls from researchers unable to read the umpteenth photocopied generation of the preprint, a sort of technical samizdat that I had to endlessly interpret,” Burnette wrote.
Burnette ultimately described his technique in Analytical Biochemistry in 1981, but by then, many scientists had incorporated western blotting into their repertoire.
Also known as the immunoblot, the western blot experiment identifies specific amino acid sequences in proteins. An antibody detects its antigen and enables a target protein to be identified in a complex protein mixture. The technique provides qualitative and semi-quantitative data about the protein.
Generally speaking, the steps are:
- Separate the macromolecules in a sample using gel electrophoresis.
- Transfer the separated molecules onto a second matrix, such as a nitrocellulose or polyvinylidene difluoride (PVDF) membrane.
- Block the matrix to prevent nonspecific binding of antibodies to the surface of the membrane.
- The transferred protein is probed using a combination of antibodies, including a primary protein specific to the protein of interest and a secondary antibody specific to the host of the primary antibody. The secondary antibody is often “complexed” with an enzyme, which will yield a detectable signal.
- Precipitates appear on the membrane, producing colorimetric changes that can be seen with the naked eye. For a more sensitive measurement, use film, a charge-coupled device camera, or fluorescently tagged antibodies
- Regardless of how results are measured, the signal’s intensity should correlate with the abundance of the antigen on the membrane.
Western Blotting Normalization
To make western blot data meaningful, it must be “normalized.” Normalization accounts for the differences of total protein in each sample that results from experimental errors.
Kenneth Oh, senior product manager at Bio-Rad Laboratories, has written extensively about the need for western blotting normalization. Oh identifies two methods to normalize data.
“The dominant western blot method, known as housekeeping protein (HKP) normalization, is difficult to use and requires careful testing to yield accurate results. However, an alternative method called total protein normalization (TPN) is available to researchers that is faster and more reliable,” Oh wrote in a recent editorial.
Single protein detection and total protein normalization are the two ways to normalize western blot data.
Single protein detection requires constraints. According to an article in Bioradiations, “positive and negative controls must be separately established, consistent protein expression levels across different samples should be tested, and a scalar linear signal response needs to be confirmed across a range of protein loading amounts.” The time-consuming process is cumbersome to achieve.
In total protein normalization, all the sample’s proteins are visualized. Their total abundance serves as the basis of normalization. While this approach represents a change from the longstanding norm, total protein normalization often yields more accurate results. The technique is accomplished using reversible staining, irreversible staining, or stain-free methods.
Legacy of the Western Blot
Since 1981, Burnette’s western blot has since been cited over 5000 times in literature. Its ability to detect specific proteins from among a mixture of proteins stands as a trustworthy way to achieve high-quality, publishable results.
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