A loading control is a fundamental tool in molecular biology experiments, functioning as an internal reference standard within a biological sample. It is typically a protein whose abundance remains stable and unaffected across all experimental conditions being tested. Scientists use this internal benchmark to verify that the amount of material initially placed into the experiment was consistent across every sample. By confirming equal sample input and processing efficiency, the loading control ensures that any observed differences in a target protein’s signal are due to a genuine biological change, not a technical error.
The Essential Need for Normalization
The necessity of using a loading control arises from the many sources of technical variation inherent in laboratory procedures. Even with precise pipetting, small inaccuracies can occur when measuring and loading protein samples onto a gel. Initial quantification of a sample’s total protein content, often done via colorimetric assays, may not perfectly reflect the amount of a specific protein that is successfully loaded.
Following sample loading, the experimental process continues to introduce potential for variability at multiple stages. The separation of proteins by size in a gel, and the subsequent physical transfer of those proteins from the gel onto a solid membrane, rarely achieve 100% efficiency across all samples. Transfer efficiency can vary slightly from lane to lane, or even be affected by the lane’s position on the edge of the gel, an issue known as the “edge effect”.
Without an internal reference point, a researcher cannot definitively conclude why one sample shows a stronger signal for a target protein than another. A stronger signal could indicate a true biological increase in the protein’s abundance due to the experimental treatment, or it could simply mean that more of that sample was accidentally loaded or transferred more efficiently. The loading control acts as a built-in monitor for these technical errors.
The loading control makes a direct comparison between samples possible through a mathematical process called normalization. Normalization involves calculating the ratio of the target protein’s signal intensity to the loading control’s signal intensity within the same sample. This ratio effectively adjusts the target protein’s signal for any differences in sample input or processing efficiency, isolating the true biological change. By accounting for technical fluctuations, normalization transforms semi-quantitative data into reliable, comparative measurements.
Criteria for Selecting Reliable Controls
An effective loading control must satisfy strict biological and chemical criteria to serve as a dependable reference point. The fundamental requirement is that the protein must be constitutively expressed, meaning its concentration remains stable and unchanged regardless of the experimental treatment or condition being studied. For instance, if an experiment investigates a drug’s effect on cell metabolism, the loading control protein cannot be one whose expression is also influenced by metabolic changes.
The loading control should also be widely and abundantly expressed across all cell types or tissues being compared. Proteins that fulfill this criterion are often referred to as housekeeping proteins because they are involved in basic cellular functions necessary for survival, such as structural maintenance or general metabolism. However, even housekeeping proteins must be validated for each specific experimental system, as their expression can sometimes be altered by factors like cell density or disease state.
A practical consideration is the molecular weight of the chosen control protein. It must possess a significantly different molecular weight from the target protein of interest to ensure the two proteins separate cleanly and appear as distinct bands on the analytical result. If the control and the target are too similar in size, their signals will overlap, making accurate and independent quantification impossible.
Finally, the signal produced by the loading control must fall within the linear dynamic range of the detection system. This means the signal intensity must be directly proportional to the amount of protein present. If the control protein is too abundant, its signal may become saturated, or “burnt out,” which prevents accurate quantification and renders the control useless for normalization.
Specific Types and Applications in Protein Analysis
The most common application for loading controls is in Western blotting, a technique used to detect specific proteins in a sample. Historically, the primary method involved using antibodies to detect specific, highly-expressed housekeeping proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Beta-actin are two frequently used examples, as they are abundant cytoplasmic proteins required for glycolysis and cell structure, respectively.
While antibody-based controls are convenient, they have limitations. For example, GAPDH expression can be altered under conditions of hypoxia, which would invalidate its use as a control in that context. Furthermore, these controls only account for a single protein, and the antibody detection process itself introduces another source of variability. The linear range of detection for a single housekeeping protein can be narrow, potentially leading to signal saturation with high protein loads.
An alternative approach that has gained widespread acceptance is total protein staining, which uses a chemical dye to stain all proteins present on the membrane. Reagents like Ponceau S or Coomassie stain the entire protein profile, providing a measurement of the total protein content in each lane instead of relying on a single protein. This method offers a wider linear dynamic range and is less susceptible to the biological regulation issues that can affect individual housekeeping proteins.
Total protein normalization is considered a more robust method for quantitative analysis because the abundance of the entire protein population is less likely to change than the level of a single protein. While Western blotting is the primary technique, the concept of an internal reference extends to other quantitative protein methods, such as mass spectrometry-based proteomics. In proteomics, reference proteins of known concentration are often spiked into the sample for normalization. The use of an appropriate internal standard, whether a single protein or total protein stain, is now standard practice and often required for publication in scientific journals.