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Western Blot Controls

By Ryan Hamnett, PhD

Western blotting involves using antibodies to selectively label proteins of interest from lysed cell or tissue samples, enabling the measurement of protein presence, size and abundance. Controls are essential for confirming the accuracy and specificity of immunoblotting, and to help identify sources of error in your experiment.

Table of Contents

Commonly used western blot controls

Control Description Purpose Notes
Positive expression control Blot alongside a lysate from a cell line or tissue sample known to express protein of interest. Confirms whether negative staining observed in the experimental sample represents a true negative result, or is a technical error. Suitable cell lines include cells that are overexpressing protein of interest. Other sources may be found on the antibody’s datasheet, in online databases, or can be found in the literature. Purified recombinant protein can also be used.
Negative expression control Blot alongside a lysate from a cell line or tissue sample that does not express protein of interest. Confirms that the staining in the experimental sample is valid, or if the primary antibody exhibits non-specific staining. Negative control samples include lysates from knockout or knockdown cell lines/organisms, or cell line/tissues known not to express target.
Loading control Blot for an additional protein, usually a from a housekeeping gene, that will not vary between different tissues and cells. Demonstrates that changes in experimental target band signal between samples is not due to variable total protein levels. See the full Loading Control guide below
Endogenous control lysate Blot alongside a lysate known to express the protein target endogenously. Similar to a positive control, demonstrates that blotting protocol was correctly performed, and indicates issues with recombinant expression. Essential when blotting recombinant protein due to issues with recombinant expression, e.g. misfolding.
No primary antibody control The primary antibody is omitted. All other protocol steps are performed as usual. Indicates non-specific staining due to the secondary antibody.
Absorption control Incubate the primary antibody with the peptide immunogen used to generate the antibody. Confirms specific binding between the primary antibody and the antigen; pre-absorption should remove genuine signal. Can give false negatives (if target epitope is similar in different proteins, pre-absorption will prevent binding to all), and false positives (if the bound immunogen non-specifically binds to lysate components).
Endogenous enzyme activity control Apply enzyme substrate, such as ECL for HRP, to membrane in the absence of antibodies. Detects inherent enzyme activity not attributable to enzyme-conjugated antibodies. Rarely implemented with WB, as harsh denaturing conditions tend to render it unnecessary. However, endogenous HRP activity has been observed in WBs in some conditions.1

Table 1:Key controls to run alongside a WB experiment.

Loading Controls

To be certain that differences in protein expression between cell or tissue samples are real and not merely due to differences in total protein concentration, blotting against consistent amounts of total protein is essential (Figure 1). Earlier protocol steps, such as quantifying protein content with a BCA, Bradford or Lowry assay, aim to ensure that a consistent quantity of protein is loaded on the gel for SDS-PAGE. Loading controls serve as a way of confirming total protein levels have stayed consistent through the whole western blotting procedure, including membrane transfer, and performing minor normalization if necessary.

Western blot witb beta-Actin loading control - Anti-Cyclin D1 Antibody [ARC0300] (A306339) - Antibodies.com

Figure 1: Beta-actin loading control validating a KO experiment. Western blot using anti-Cyclin D1 antibody in WT and Cyclin D1 knockout HeLa cells. No band is observed for cyclin D1 in the right-hand lane, but total protein levels are unaffected as confirmed by the beta-actin loading control. Therefore, the knockout of cyclin D1 has been successfully verified.

Note that to be a true loading control, the control must be blotted for on the same membrane and at the same time as the target antigen. Blotting for a loading control on a membrane that has been processed in parallel (e.g. loading the same samples into two separate gels, then running the SDS-PAGE, membrane transfer and blotting in parallel) would more accurately be referred to as a sample processing control due to potential differences in initial loading.

Which proteins are used as loading controls?

The loading control antigens tend to be housekeeping proteins: abundantly and ubiquitously expressed proteins that are involved in the essential, basic maintenance and function of all cell types, and which are therefore unlikely to vary in expression levels between cell lines and tissues.

Considerations for choosing loading controls

  1. Molecular weight (MW): A loading control protein should be a different MW to your protein of interest. This will make it possible to distinguish the control band from the target band during simultaneous blotting.
  2. Abundant expression: Ensure that the loading control is expressed at a high level in the organism, tissue, cell line, and even organelle under investigation.
  3. Experimental treatments: Loading controls should not be affected by any treatment or genetic manipulation being performed, so understanding the normal function of a loading control and checking the literature is important to understand if this is likely for a given experimental variable (Figure 2).
  4. Comparing across sample types: If an experiment involves comparing levels of a protein between tissue types, different cell lines, or different species, ensure that the chosen loading control is expressed at comparable levels across all of them to avoid misinterpretation of results.
  5. Saturation: Loading controls being abundantly expressed has a flip-side, which is that their signal can become easily saturated during detection, which would render it unsuitable for normalization. Perform an antibody dilution series to optimize antibody concentration and test a range of exposure times to find a good signal-to-noise ratio for the loading control while still avoiding saturation.
  6. Circadian time: Housekeeping genes, and indeed total protein synthesis2, can vary in abundance with circadian time, so it is important to take this into account if an experiment requires processing samples at different times of day.
  7. Fluorescence: For a multiplexed fluorescent western blot, the brightest fluorophore should be used for the protein of interest, while the dimmest fluorophore should be used for the highly abundant loading control.
Western blot witb beta-Actin loading control - Anti-Lamin B1 Antibody (A13736) - Antibodies.com

Figure 2: Loading controls can be affected by experimental conditions.Western blot using anti-Lamin B1 antibody, which can act as a nuclear loading control. However, lamin B1 is cleaved during apoptosis3, caused here by staurosporine addition, which would make it unsuitable for all experiments. Instead, beta-actin is used as a loading control.

Commonly used loading controls

Subcellular localization / Sample type Control name Molecular weight, kDa
Whole cell/cytoplasmic alpha-Tubulin 55
beta-Actin 43
beta-Tubulin 55
Cofilin 19
Cyclophilin A 18
Cyclophilin B 18
GAPDH 37
Vinculin 116
Nuclear HDAC1 62
Histone H1 15
Histone H2B 15
Histone H3 15
Lamin B1 66
PCNA 29
TATA binding protein (TBP) 38
Mitochondrial COX IV 17
Hsp60 60
VDCA1 31
Plasma membrane beta-Catenin 86
CD44 82
Na+/K+ ATPase alpha-1 112
Muscle SDHA 73
Serum Transferrin 77

Table 2:Loading controls to use in WB experiments

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