Loading Controls

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Loading Controls Handbook

Download and View Novus’ Loading Control Handbook to learn more about loading controls, how to use them with Western blots, and tips and considerations.

Loading Controls

What is a loading control?

  • Loading Control: A loading control is an antibody specific for a ubiquitously and constitutively expressed protein and can be used to normalize protein levels in Western blot. Loading control antibodies help in assessing that samples have been loaded equally across a gel. By serving as internal positive controls, they also show that reagents are functioning properly and efficient protein transfer to the membrane has occurred. Loading controls can be general whole cell proteins or specific markers for subcellular fractions (e.g. nucleus, mitochondria, membrane).

Loading Control Cell

What should I consider when choosing a loading control for my experiment?

  • Detection Size: Choosing a loading control that is close in size, but still large or small enough to be easily distinguished from your target protein is crucial. If the target protein and loading control are the same size, visualization will be impaired and data interpretation compromised.

  • Strong Expression: Ensure the chosen loading control demonstrates strong expression in your sample. Most common loading control targets are highly expressed constitutive genes required for basic cellular processes and cellular vitality (housekeeping genes). However, the expression of some rarer controls may not be as ubiquitous.

  • Expression Factors: Choose a loading control whose expression is not affected by an experimental variable (e.g. if studying a metabolic process use of GAPDH as a loading control is not recommended due its role in glycolysis). An optimal loading control should detect a globally expressed protein that is not differentially regulated in normal and disease states.

  • Direct vs Indirect Detection: Consider if the loading control antibody is suitable for direct conjugation or requires the addition of a conjugated secondary antibody for indirect detection of the loading control. With direct detection, primary antibodies are conjugated directly to an enzyme, like HRP, for chemiluminescent detection or to fluorochromes including Alexa Fluor® dyes, Janelia Fluor® dyes or DyLight® dyes for fluorescent Western blotting.

When do I need to use a loading control antibody?

  • Quantitative Comparisons: Loading controls are necessary when comparing signal between wells. Observed changes in target protein expression can only be considered true if expression of the loading control is equal across wells.

  • Loading and Transfer Confirmation: Loading controls confirm all samples have been loaded and transferred equally across wells. Uneven transfer from the gel to membrane can be determined by analysis of loading control signal.

  • Publishing your study: Most journals require loading control data in western blot analysis to ensure differential expression between experimental samples is real. Loading controls normalize target protein expression across all wells.



View All Loading Control Antibodies




Common Loading Controls

Subcellular Localization Loading Controls Molecular Weight (kDa)
Cytoplasm/Whole Cell

Vinculin

Alpha-tubulin

beta Tubulin

Actin

beta-Actin

GAPDH

Cyclophilin B

Cofilin

116

55

55

45

43

37

21

18

Membrane

Sodium Potassium ATPase Alpha 1

beta-Catenin

CD44

112

86

82

Mitochondria

HSP60

VDAC1/porin

COX IV

60

31

17

Nucleus

Lamin B1

HDAC1

PCNA

Histone H3

66

60

28

17

Serum Transferrin 77


Western Blot showing lysates from HeLa parental cell line and LC3B knockout HeLa cell line either untreated or treated with Chloroquine and probed for LC3B Antibody. Western Blot depicting lysates from human brain, mouse brain, and rat brain tissue probed for GAPDH Antibody with specific band expression at 39 kDa.
Knockout Validated Western Blot showing lysates of HeLa parental cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM Chloroquine. Membrane was probed with 2.0 µg/mL of Rabbit Anti LC3B Monoclonal Antibody (1251A) (NBP2-46892) followed by HRP-conjugated Secondary Antibody (HAF008). A specific band was detected for LC3B at a molecular weight of approximately 15 kDa in the parental HeLa cell line, but is not detectable in the knockout cell line. GAPDH is shown as a loading control, remaining unchanged. Western blot highlighting GAPDH expression in lysates of human brain, mouse brain, and rat brain tissue. Membrane was probed with 0.05 µg/mL of Mouse Anti- GAPDH/G3PDH Monoclonal Antibody (MAB5718) followed by HRP-conjugated Secondary Antibody (HAF007). A specific band was detected for GAPDH/G3PDH at approximately 39 kDa.


Western Blot showing brain tissue lysates from control and diabetic rats treated with insulin receptor inhibitor IOMe or Nigella sativa oil (NSO) and probed with various insulin induced signaling molecules including p-IRS1, p-AKT, p-GSK-3β, p-Tau, and PP2A-alpha, with AKT1, GSK-3β, and β-actin as normalization loading controls.
Representative western blotting analyses of brain insulin-induced signaling molecules. Protein level profile of p-IRS1-Tyr612 was normalized to the total amount of β-actin (NB600-501) protein and p-AKT1-Ser473 level was normalized to the total amount of AKT1 (NBP2-01724). p-GSK3β-Ser9 was normalized to the total amount of GSK3β (MAB2506). p-Tau-Ser356 and PP2A-alpha (MAB1653) levels were normalized to the total amount of β-actin (NB600-501) protein in the brain tissue of diabetic (D) rats injected with insulin receptor inhibitor IOMe and treated with NSO (Nigella sativa oil). Image collected and cropped by CiteAb from the following publication:
(https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172429) licensed under a CC-BY license.




Alternatives to Loading Controls

Total protein quantification assays can be performed as an alternative to traditional loading controls. A quick and reversible membrane stain such as Ponceau S Staining Solution (Cat. No. 5225) can be applied to visually confirm equal lane loading and successful transfer of proteins to the membrane. Total protein assay is also utilized in Simple Western systems. Simple Westerns are automated Western blot instruments that allow for faster, more reproducible detection and characterization of proteins. With instruments like Jess, you can perform total protein normalization assays at the same time as your immunoassay.


Simple Western lane view of protein normalization showing total protein membrane stain with 14-3-3 antibody as a loading control. Simple Western lane view of protein normalization visualized as the traditional total protein membrane stain with 14-3-3 antibody used as a loading control
(Left) Knockout Validated Western blot showing lysates from U20S parental cell line and Dynamin knockout cell line (KO). Membrane was probed with Mouse Anti-Dynamin Monoclonal Antibody (NB110-60491) at a 1:1000 dilution, followed by HRP-conjugated Secondary Antibody and detected with ECL reagent. A specific band was detected for Dynamin-1 (as indicated) in the parental U20S cell line but is not in the KO U20S cell line. The Ponceau stained transfers of each blot are shown to confirm equal protein loading. Data image, protocol, and testing courtesy of YCharOS Inc. (Right) Simple Western lane view of protein normalization visualized as the traditional total protein membrane stain with 14-3-3 antibody used as a loading control.

Learn more about Simple Western






Loading Control Tips and Considerations

Target Protein Note
Beta-actin Not suitable for nuclear extract as beta-actin is a component of chromatin remodeling complexes (1). May not be suitable for studies involving subjects with a large age difference (2).
GAPDH Not suitable for oxygen-related studies as hypoxia can upregulate GAPDH expression (3). May not be suitable for studies involving subjects with a large age difference (2).
Alpha-tubulin May not be suitable for studies involving subjects with a large age difference (2). Tubulin expression can be affected by anti-cancer and anti-fungal drugs.
Lamin B1 Not suitable for embryonic stem cells (4).
COX IV Many proteins run at around 15~17 kDa; hence, it may be necessary to consider an alternative control antibody if your protein of interest is similar in size to COX 4.
Transferrin Transferrin levels can be influenced by some disease states and treatments such as retinoic acid.
Histone H3 Many proteins run at around 15~17 kDa; hence, it may be necessary to consider an alternative control antibody if your protein of interest is similar in size to Histone H3.

Suggested References:

  1. Olave IA et al. Nuclear actin and actin-related proteins in chromatin remodeling. Annu Rev Biochem. 2002;71: 755-81 [PMID: 12045110]
  2. Vigelsø A et al. GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle. J Appl Physiol (1985). 2015 Feb 1;118(3):386-94 [PMID: 25429098]
  3. Zhong H and Simons JW. Direct comparison of GAPDH, beta-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. Biochem Biophys Res Commun. 1999 Jun 16;259(3):523-6 [10364451]
  4. Kim Y et al. Mouse B-type lamins are required for proper organogenesis but not by embryonic stem cells. Science. 2011 Dec 23;334(6063):1706-10 [PMID 22116031]


Alexa Fluor® is a registered trademark of Molecular Probes, Inc, a Thermo Fisher Scientific Company.

DyLight® is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.

Janelia Fluor® Dyes sold under license from the Howard Hughes Medical Institute, Janelia Research Campus.