Loading Controls

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Generate Publication Quality Western blots

A western blot provides a readout of differences in protein expression levels, for example changes in protein levels over a certain time period, differences in knock-out versus wild type cell lines, and altered expression in patient samples. However, to draw conclusions from a western blot, you need to ensure that the observed differences are only due to altered protein expression levels rather than gel loading or protein transfer errors. This is where western blotting loading controls can help.

Table 1 shows the molecular weight and cellular localization of the commonly used housekeeping proteins. Click on your housekeeping protein of interest to find out more information on the antibodies that Bio-Rad offers and select the housekeeping protein antibody that is right for your experiment.

Table 1. Common housekeeping proteins.

*The exact molecular weight might vary from species to species

What Are Loading Controls?

• Antibodies that detect a highly expressed protein in a certain cell or sample type
• Encoded by housekeeping genes, housekeeping proteins are proteins required for the maintenance of basic cell functions and are often ubiquitously and abundantly expressed. Expression levels of some housekeeping proteins may vary with cell type and treatment (Hagner-McWhirter 2015) 
• Tools for normalizing gel loading differences and western blot quantification (by software programs such as ImageJ)

Why Are Loading Controls Crucial?

• Loading controls are particularly necessary when signal levels of modified proteins are compared between samples
• They ensure you have loaded and transferred equal amounts of protein across all wells of your western blot
• Journals now require appropriate controls to be used in your experimental setup

Choosing and Using Loading Controls

1. Ensure your protein of interest and loading control have different molecular weights. This allows you to easily distinguish bands representing your protein of interest from those representing your housekeeping protein.
2. Ensure your loading control is highly expressed in your sample and its level remains unchanged throughout an experiment. Don’t use a housekeeping gene that you’ve found to vary in expression level during your experimental treatment previously.
3. Probing with a loading control can be carried out alongside the target antibody by cutting the membrane before antibody incubation, or after the blot has been stripped of previously bound antibodies.
4. Perform titration experiments on your samples to determine the concentration of antibody against your housekeeping protein and blot exposure time that gives optimum signal-to-background ratio without signal saturation. Housekeeping proteins are often highly expressed, making it easy for the signal from the housekeeping protein detection to become saturated and affect normalization.
5. If you are performing a multiplexed fluorescent western blot experiment, choose your brightest fluorophore for your protein of interest and your dimmest fluorophore for your housekeeping protein. This will prevent overexposure and therefore oversaturation of the signal from your housekeeping protein.

Common Loading Controls

Beta Actin

Beta Actin is a 42 kDa protein ubiquitously expressed in all eukaryotic cells. It plays a critical role in the regulation of cell migration and gene expression through control of the cellular G-actin pool (Bunnell et al. 2011). Beta actin is commonly used as a western blotting loading control because it is ubiquitously expressed to high levels and is stable under most experimental conditions. However, beta actin may not be suitable for use in samples from tissues containing high actin levels, like skeletal muscle, due to potential oversaturation of signal.

Cofilin 1

Cofilin 1 is a widely distributed intracellular actin-modulating protein that binds and depolymerizes filamentous F-actin and inhibits the polymerization of monomeric G-actin in a pH-dependent manner. It is involved in the translocation of the actin-cofilin complex from cytoplasm to nucleus (Xiang et al. 2012).

GAPDH

GAPDH is a 36 kDa protein which catalyzes the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate, a crucial step in carbohydrate metabolism. GAPDH has been shown to translocate to the nucleus under oxidative stress conditions, whereby it mediates cell death. GAPDH can bind to several proteins that are responsible for neurodegenerative diseases, such as amyloid precursor protein and Huntingtin (Hara et al. 2006). It is highly expressed in most tissues.

HSP60

Heat shock protein 60 (HSP60), also known as 60 kDa chaperonin, HSPD1, HSP60, GROEL, and mitochondrial matrix protein P1, is a member of the chaperonin family. HSP60 is a mitochondrial protein essential for the folding and assembly of newly imported proteins in the mitochondria (Kondoh and Osada 2013). Note, expression levels of HSP60 may change upon oxidative stress or temperature stress. Tumor cells can also display increased levels of HSP60.

Lamin B1

Lamin B1 is one of the two B type lamin proteins. The lamin family of proteins make up the lamina matrix, a two-dimensional matrix of proteins located next to the inner nuclear membrane, and are highly conserved in evolution. Lamin proteins are thought to be involved in nuclear stability, chromatin structure, and gene expression. During mitosis, the lamina matrix is reversibly disassembled as the lamin proteins are phosphorylated. Duplication of the gene encoding Lamin B1 has been linked to autosomal dominant adult-onset leukodystrophy (ADLD) (Finnsson et al. 2015).

Lamin B1 is commonly used as a loading control due to the fact that B type lamins are present in every cell type and are highly conserved across species. However, Lamin B1 protein is not suitable as a loading control for samples that do not contain a nuclear envelope.

PCNA

Proliferating cell nuclear antigen (PCNA), also known as cyclin, is 29 kDa nuclear protein that plays a crucial role in DNA synthesis (Li et al. 1995) through its interaction with FEN1 (Wu et al. 1996). PCNA also acts as the auxilliary protein for DNA polymerase δ (Bravo et al. 1987).

PCNA is a popular choice as a loading control as it is highly conserved between mammalian species and other vertebrates. However, note, that PCNA is rapidly degraded upon activation of DNA damage pathways.

Transferrin

Transferrin is a 79 kDa iron binding glycoprotein that plays an important role in the transportation of iron from the intestine, reticuloendothelial system, and liver parenchymal cells to all proliferating cells in the body (Yang et al. 1984, Kawabata, 2019).

The expression of transferrin is affected in some inherited diseases and by retinoic acid treatment. Expression levels of transferrin are also higher under conditions of iron deficiency and lower in liver disease.

VDAC1

VDAC1 is a 32 kDa voltage-dependent anion channel protein located in the mitochondrial membrane and outer cell membrane. VDAC1 facilitates the transportation of ATP and other ions across the outer mitochondrial membrane to the cytoplasm. It is involved in the regulation of cell volume (Reina et al. 2010).

When using VDAC1 as a loading control, note that VDAC1 may be highly expressed in tumor cells and can oligomerize in apoptotic cells.

References

• Bravo R et al. (1987). Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature 326, 515–517.
• Bunnell TM et al. (2011). β-Actin specifically controls cell growth, migration, and the G-actin pool. Mol Biol Cell 22, 4,047–4,058.
• Hagner-McWhirter A et al. (2015). Cy5 total protein normalization in western blot analysis. Anal Biochem 486, 54–61.
• Hara MR et al. (2006). GAPDH as a sensor of NO stress. Biochim Biophys Acta 1,762, 502–509.
• Kawabata H (2019). Transferrin and transferrin receptors update. Free Radic Biol Med 133, 46–54.
• Li X et al. (1995). Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen. J Biol Chem 270, 22,109–22,112.
• Kondoh Y and Osada H (2013). High-throughput screening identifies small molecule inhibitors of molecular chaperones. Curr Pharm Des 19, 473–492.
• Reina S (2010). Swapping of the N-terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti-aging features to the cell. FEBS Lett 584, 2,837–2,844.
• Wu X et al. (1996). Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA. Nucleic Acids Res 24, 2,036–2,043.
• Xiang Y et al. 2012. Cofilin 1-mediated biphasic F-actin dynamics of neuronal cells affect herpes simplex virus 1 infection and replication. J Virol. 86, 8,440–8,451.
• Yang F et al. (1984). Human transferrin: cDNA characterization and chromosomal localization. Proc Natl Acad Sci USA 81, 2,752–2,756.