Loading controls serve as a vital method of verifying the results acquired through western blotting. Loading controls are antibodies that are used to detect proteins within samples. When western blots are used to determine the levels of protein expression in a sample, loading controls ensure that the results aren't due to loading or protein transfer errors. When using a loading control, the right type of control must first be identified.
The Benefits of Using a Loading Control
Loading controls convey numerous benefits, generally regarding quality. Without a loading control, western blots cannot be published because they are not sufficiently verified. Because of this, loading controls are entirely essential for research that may eventually be submitted for publication. With a loading control, common errors such as failed transfers can be almost immediately distinguished. This reduces the amount of time that might be spent on inconclusive or incorrect results.
Further, issues such as the "edge effect" -- when proteins in outer lanes drift towards the center -- can be removed through the use of a loading control, fine-tuning the entire blotting process. Loading controls can be further used to minimize the impact of erratic protein expression levels. In any situation in which proteins may be affected by either loading or protein transfer errors, loading controls are even more vital.
The Most Popular Types of Loading Control
There over a dozen popular types of loading control. Depending on the western blot itself, any of these proteins may be ideal. Here are a variety of the most popular forms of loading control.
- Actin. Actin is ideal for whole cell and cytoplasmic extract applications. With a variety of forms available, Actin is an extremely versatile loading control. Actin cannot be used on skeletal muscle samples, as it is a major component in muscle action.
- GAPDH. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is another whole cell and cytoplasmic extract loading control. Breaking down glucose and carbon molecules, GAPDH serves as a glycolysis catalyst.
- Porin. Porin is an ideal loading control for mitochondrial proteins. Porin is a voltage-dependent anion-selective channel protein, which is found in a variety of tissues.
- Lamin B1. Suitable primarily for nuclear protein envelopes, Lamin B1 is theoretically believed to be responsible for some element of nuclear stability within the cell. Lamin B1 should not be used as a loading control if the nuclear envelope isn't available.
- TBP. TATA binding protein (TBP) is another loading control for nuclear proteins. A transcription binding protein, TBP works directly with RNA polymerase II and is found within DNA sequencing.
Not only are there different types of loading control, but loading controls are also available in different formats supplied unconjugated or conjugated to a specific label, enzyme or dye for simplified detection.
The Drawbacks of Using a Loading Control
As with many accuracy checks, a loading control can increase the cost and time associated with each western blot. Whether or not this is worthwhile depends on the laboratory and their needs. Because loading controls have to be purchased, the exact cost of utilizing a loading control will be highly dependent on where the loading controls are sourced and how much they cost to purchase. Further, using the wrong loading controls for a specific application can potentially cause some issues regarding the accuracy and production of the result. Nevertheless, loading controls are virtually a requirement for reliable testing.
Labs that are not familiar with the use of loading controls may want to further investigate their laboratory needs first -- and the right type of loading controls for them.
