Loading controls are commonly used in gel electrophoresis techniques, such as western blotting, to verify that the gel lanes have been evenly loaded with sample material, and they are typically used to standardize the results from these studies. Since the proteins in loading controls are abundantly expressed, they also allow investigators to determine if there is an even transfer across the entire gel. A loading control is absolutely required when western blots are prepared for publication.
Proteins Used As Loading Controls
Origin: Whole Cell / cytoplasmic
Molecular weight (kD): 43
Beta actin is one of six isoforms of actin. Actin is a globular, roughly 42-kDa protein found in all eukaryotic cells (except for nematode sperm) where it may be present at concentrations of over 100 microM. It is also one of the most highly-conserved proteins, differing by no more than 20% in species as diverse as algae and humans. It is the monomeric subunit of microfilaments, one of the three major components of the cytoskeleton, and of thin filaments, which are part of the contractile apparatus in muscle cells. Thus, actin participates in many important cellular functions, including muscle contraction, cell motility, cell division and cytokinesis, vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. This loading control is not suitable for skeletal muscle samples. Changes in cell-growth conditions and interactions with extracellular matrix components may alter actin protein synthesis (Farmer et al, 1983).
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
Origin: Whole Cell / cytoplasmic
Molecular weight (kD):30-40
Because the GAPDH gene is often stably and constitutively expressed at high levels in most tissues and cells, it is considered a housekeeping gene. For this reason, GAPDH is commonly used by biological researchers as a loading control for western blot and as a control for RT-PCR. However, many researchers report different regulation of GAPDH under specific conditions. Consequently, the use of GAPDH as loading control has to be carefully evaluated.
Whole Cell / cytoplasmic
Molecular weight (kD):55
Tubulin is one of several members of a small family of globular proteins. The most common members of the tubulin family are alpha-tubulin and beta-tubulin, the proteins that make up microtubules. Each has a molecular weight of approximately 55 kiloDaltons. Microtubules are assembled from dimers of alpha- and beta-tubulin. These subunits are slightly acidic with an isoelectric point between 5.2 and 5.8. Tubulin expression may vary according to resistance to antimicrobial and antimitotic drugs (Sangrajrang S. et al, 1998, Prasad V et al, 2000)
Molecular weight (kD): 31
Porins are beta barrel proteins that cross a cellular membrane and act as a pore through which molecules can diffuse. Unlike other membrane transport proteins, porins are large enough to allow passive diffusion – i.e., they act as channels that are specific to different types of molecules. They are present in the outer membrane of Gram-negative bacteria, the mitochondria, and the chloroplast.
Cytochrome C Oxidase (COXIV)
Molecular weight (kD):16
Cytochrome C Oxidase (COXIV) is located in the inner mitochondrial membrane and serves as the terminal enzyme complex of the mitochondrial electron transport chain. COXIV collects electrons that are transferred from reduced cytochrome C and donates them to molecular oxygen, resulting in reduction to water. COXIV is expressed at a consistently high level and thus makes a good mitochondrial loading control; however, many proteins run at the same size.
Origin: Nuclear envelope
Molecular weight (kD): 66
The nuclear lamina consists of a two-dimensional matrix of proteins located next to the inner nuclear membrane. The lamin family of proteins make up the matrix and are highly conserved in evolution. During mitosis, the lamina matrix is reversibly disassembled as the lamin proteins are phosphorylated. Lamin proteins are thought to be involved in nuclear stability, chromatin structure and gene expression. Vertebrate lamins consist of two types, A and B. This gene encodes one of the two B type proteins, B1.This protein is not suitable for samples where the nuclear envelope has been removed.
TATA binding protein TBP
Molecular weight (kD):38
The TATA binding protein (TBP) is a transcription factor that binds specifically to a DNA sequence called the TATA box. This DNA sequence is found about 25-30 base pairs upstream of the transcription start site in some eukaryotic gene promoters. TBP, along with a variety of TBP-associated factors, make up the TFIID, a general transcription factor that in turn makes up part of the RNA polymerase II preinitiation complex. As one of the few proteins in the preinitation complex that binds DNA in a sequence-specific manner, it helps position RNA polymerase II over the transcription start site of the gene. However, it is estimated that only 10-20% of human promoters have TATA boxes. Therefore, TBP is probably not the only protein involved in positioning RNA polymerase II. This protein is not suitable for samples where the nuclear envelope has been removed.