Nuclear factor erythroid 2-related factor 2, commonly known as Nrf2, functions as a transcription factor within cells. This protein plays a role in helping cells manage stress, particularly from oxidative damage. For scientists investigating Nrf2, understanding its physical characteristics, such as its molecular weight, is foundational for developing experiments and interpreting results, and assists in correctly identifying the protein.
Predicted vs. Observed Molecular Weight
The predicted molecular weight of human Nrf2, based on its amino acid sequence, is approximately 65 to 68 kilodaltons (kDa). This calculated size reflects the protein’s direct composition of amino acids. However, when Nrf2 is analyzed in laboratory experiments, such as a Western blot, its observed molecular weight often appears significantly higher. Researchers typically detect Nrf2 as a band ranging from 95 to 110 kDa, a notable discrepancy from its predicted size.
Some antibody data sheets have even incorrectly labeled the higher band as “nonspecific” due to a lack of awareness regarding Nrf2’s true apparent weight. This disparity emphasizes that theoretical predictions alone do not always fully represent a protein’s behavior in a cellular environment, and the biologically relevant form of Nrf2 is generally accepted to migrate in the 95-110 kDa range under denaturing conditions.
Factors Influencing Nrf2’s Apparent Weight
The primary reason Nrf2’s observed molecular weight is greater than its predicted size is due to post-translational modifications (PTMs). PTMs are chemical tags or changes added to a protein after it has been synthesized. These additions can increase the protein’s overall mass and affect how it migrates during laboratory separation techniques.
Common PTMs that influence Nrf2’s apparent weight include phosphorylation and ubiquitination. Phosphorylation involves the addition of phosphate groups, while ubiquitination attaches ubiquitin molecules. Both types of modifications add mass to Nrf2, making it appear heavier in experiments. Other modifications like acetylation, SUMOylation, and glycosylation can also contribute to the altered molecular weight and influence Nrf2’s stability and function. Additionally, Nrf2 exists as multiple isoforms, which are slightly different versions of the protein with varying molecular weights.
Experimental Determination of Molecular Weight
Scientists routinely determine the observed molecular weight of Nrf2 using a technique called SDS-PAGE, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method separates proteins based on their size by pushing them through a gel matrix using an electric current. Smaller proteins move more quickly through the gel, while larger proteins move more slowly.
After proteins are separated by SDS-PAGE, Western blotting is then used to specifically identify Nrf2. In Western blotting, the separated proteins are transferred from the gel onto a membrane. Specific antibodies designed to recognize Nrf2 are then applied, allowing researchers to visualize the protein and estimate its size by comparing its migration distance to a ladder of known molecular weight markers. This combined approach provides the experimental evidence for Nrf2’s observed molecular weight, highlighting the influence of post-translational modifications on its migration.