Loading Controls for Western Blot

What Is a Loading Control?

A Western blot loading control is an antibody that specifically detects a constitutively expressed protein. Loading control antibodies help confirm that equal amounts of sample have been loaded in each lane across the gel. Loading controls serve as internal positive controls. They can be used to normalize protein levels between lanes of the Western blot, demonstrate that detection reagents are functioning properly, and confirm that proteins have been efficiently transferred to the membrane. Loading control antibodies can be selected to recognize housekeeping proteins in whole cell lysates or marker proteins for subcellular fractions (e.g. nucleus, mitochondria, or membrane).

View Loading Control Antibodies

Loading control antibodies in different cell organelles

Common Loading Controls
What Should I Consider When Choosing a Loading Control?
When Do I Need to Use Them?
Data Examples
Alternatives to Loading Controls
Tips and Considerations
Additional Western Blot Resources
References

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 COX 4	60 31 17
Nucleus	Lamin B1 HDAC1 PCNA Histone H3	66 60 28 17
Serum	Transferrin	77

What Should I Consider when Choosing a Loading Control for My Experiment?

Size of Recognized Protein: Choosing a loading control whose target protein is close in size, but still easily resolved from your protein of interest is crucial. If these proteins are nearly the same size, band visualization and data interpretation will be compromised.

Strong Expression: Select a loading control antibody that detects a highly expressed protein in your sample. Most common loading control targets are highly and constitutively expressed because they are required for basic cellular processes (housekeeping genes).

Expression Factors: Choose a loading control whose expression is not affected by experimental variables (e.g. if studying a metabolic process, the use of GAPDH as a loading control is not recommended due its role in glycolysis). An optimal loading control antibody should detect a 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 if the use of a secondary antibody for indirect detection of the target is more suitable. With direct detection, primary antibodies are conjugated directly to an enzyme, such as HRP, for chemiluminescent detection or to fluorochromes such as Alexa Fluor^®, Janelia Fluor^®, or DyLight^® dyes for fluorescent Western blotting. See our Explaining Antibody Conjugation blog to learn more.

When Do I Need to Use a Loading Control Antibody?

Quantitative Comparisons: Loading controls are necessary when comparing signals between lanes on the blot. Observed changes in target protein expression can only be considered valid if expression of the loading control target is equivalent 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 the membrane can be determined by analysis of loading control's signal.

Publishing Your Study: Most journals require loading control data in Western blot analyses to ensure differential expression between experimental samples is not an result of uneven sample loading.

Knockout validated Western blot showing lysates of parental and LC3B knockout (KO) HeLa cell lines untreated (-) or treated (+) with 50 μM Chloroquine (Catalog # 4109). Membrane was probed with 2.0 µg/mL of LC3B Antibody (1251A) (Catalog # NBP2-46892) followed by HRP-conjugated Secondary Antibody (Catalog # HAF008). A specific band was detected for LC3B at approximately 15 kDa in the parental HeLa cell line, but not in the KO cell line. GAPDH is shown as a loading control, remaining unchanged.

Western blot highlighting GAPDH expression in lysates of brain tissue from human, mouse, and rat. Membrane was probed with 0.05 µg/mL Mouse GAPDH/G3PDH Antibody (Catalog # MAB5718) followed by HRP-conjugated Secondary Antibody (Catalog # HAF007). A specific band was detected for GAPDH/G3PDH at approximately 39 kDa.

western blotting analyses of brain insulin-induced signaling molecules

Representative Western blot analysis of insulin-induced signal transduction molecules in brain tissue of diabetic (D) rats injected with insulin receptor inhibitor IOMe and treated with NSO (Nigella sativa oil). Protein level profile of phospho-IRS1-Tyr612 was normalized to the total amount of β-Actin (Catalog # NB600-501); phospho-AKT1-Ser473 level was normalized to the total amount of AKT1 (Catalog # NBP2-01724); phospho-GSK3β-Ser9 was normalized to the total amount of GSK-3β (Catalog # MAB2506); phospho-Tau-Ser356 and PP2A-alpha (Catalog # MAB1653) levels were normalized to the total amount of β-Actin (Catalog # NB-600-501). Image collected and cropped by CiteAb from Balbaa, M. et al., PLoS ONE May 15; 12(5):e0172429., licensed under a CC-BY license.

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 (Catalog # 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.

Learn More About Simple Western

Knockout validated Western blot showing lysates from parental and Dynamin knockout (KO) U20S cell lines. Membrane was probed with Dynamin Antibody (Catalog # NB110-60491) at a 1:1000 dilution, followed by HRP-conjugated Secondary Antibody and ECL reagent. A specific band was detected for Dynamin-1 in the parental cell line but not in the KO cell line. The Ponceau stained transfers of each blot confirm equal protein loading between lanes. Data image, protocol, and testing courtesy of YCharOS Inc.

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.

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 4	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.