How do I choose a loading control for my western blot?

Think about the last time you generated western blot data. Did you find the predicted differences in signal among your experimental samples, or were you left wondering what went wrong when you saw no change in protein levels? Here’s one more question: Did you remember to probe for a reliable loading control protein?

Many of us default to classic cytoskeletal or housekeeping proteins, never thinking twice about loading control western blots. However, there are a number of reasons why this often-overlooked blot deserves further attention.

What are loading controls and why are they important?

A loading control is a protein that is highly and constitutively expressed in your sample and that is used as a positive control in your western blot experiment. The most important reason for using a loading control is to ensure the reliability of your data and conclusions, as it helps demonstrate that any differences in protein expression are due to experimental conditions and not variations in sample loading or membrane transfer. If your samples are loaded unevenly across a gel or membrane transfer is inconsistent, you won’t know if your experiment supports or rejects your hypothesis.

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Typical loading controls include antibodies for proteins such as GAPDH, β-actin, α-tubulin, histone 3, vinculin and other common housekeeping proteins that are required for basic cell function. These proteins are typically ubiquitously expressed in most sample types and their abundance is usually not affected by biological variations. However, depending on your experimental conditions, different antibodies may be more appropriate, so it’s worth thinking critically about loading control selection.

When choosing a loading control, there are three key criteria for consideration:

1. The protein level of the loading control should not be altered by experimental conditions. 

2. The loading control and protein of interest should be easily distinguishable from each other by western blot.

3. The signal detected for both the loading control and the protein of interest should be in a linear range.

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So, how can you address all of these principles when preparing to run a western blot for a loading control protein? Find out how to prevent and resolve some common issues below. You’ll also find reference tables for loading control proteins organized by molecular weight, sub-cellular localization, and host species. Be sure to bookmark this page for the next time you need to choose a loading control.

1. The protein level of the loading control should not be altered by experimental conditions.

The first step in choosing a loading control is ensuring that the loading control protein of choice is appropriate for the experiment being performed. It is worth taking into account where the target protein is expressed and if there is a loading control protein that is also expressed in the same sub-cellular location. For example, if you have prepared lysates consisting solely of nuclear proteins, it would be more applicable to normalize against a DNA-binding protein than a cytoskeletal protein.

Western blot analysis of extracts from various cell lines using TBP (D5C9H) XP^® Rabbit mAb #44059.

In addition to where a protein is expressed, it's also important to understand how that expression can be altered as a result of your experimental conditions. Let’s say you have quantified the total protein in your samples and are confident you have loaded the same amount across your gel. However, when you go to image, you find significantly different signal in your experimental samples than in your control samples.

Related Resource: Western Blot Troubleshooting Guide

What went wrong? It could be that the loading control protein levels are affected by the experimental conditions. In most cases, a quick literature search before choosing a loading control can help confirm if any shifts in expression are expected for the normalization control protein of choice.

2. The loading control and protein of interest should be easily distinguishable from one another by western blot.

Sometimes, you’re up against a deadline, have limited resources (like a precious sample), or simply want to plan the most efficient experiment possible. In those instances, you may consider co-incubating the loading control antibody with the target protein antibody. Now, before you start mixing antibodies, it is crucial to think about what those results will look like and how you can maintain the integrity of your data, all while being efficient.

Consider Molecular Weight to Avoid Compounding Signals When Co-Incubating Antibodies

One way to avoid compounding signals is to choose a loading control protein with a very different molecular weight than your protein of interest. By doing so, both primary antibodies can be co-incubated and the signal for each target would appear at different positions on the same blot. 

The table below shows the molecular weight and cellular localization of some proteins commonly used as loading controls:

Table 1: Common loading control proteins and their cellular localization and molecular weight (kDa). 

What do you do if your protein of interest has a similar molecular weight as the loading control protein you want to detect? Easy! Just select antibodies that are produced in different host species. The host species can be found on every CST^® antibody datasheet and web page by looking up the Source under the Supporting Data section.

If an antibody for detecting the protein of interest is produced in rabbits, you can use a loading control antibody produced in mice and co-incubate the membrane with both primaries. After washing, you can incubate with an anti-rabbit secondary antibody, develop, and take images of bands showing the protein of interest. Another wash step later, and the blot can be incubated with an anti-mouse secondary antibody. Wash, develop, and image, just like before, and voila! This time, you have captured bands representing the loading control protein.

The table below shows CST antibodies for common loading control antibodies by host species:

Table 2: Common loading control antibodies from CST by host species.

One final option for preventing overlapping signals between the target protein and loading control protein is to employ both strategies—use a loading control antibody that recognizes a protein at a very different molecular weight than the protein of interest and is produced in a different host species. Doing so minimizes any risk of confusion over which band represents which protein.

Related Blog: Loading Control Expression in Mouse Tissues

Each of these strategies not only saves time and reagents, but also saves you from having to run duplicate gels with a precious sample. Plus, you’re sure to get bonus points for demonstrating consistent gel loading across samples, and for making quantitative analysis easier, all with a single blot. Just remember to take multiple exposures to capture the optimal signal for both proteins.

3. The signal detected for both the loading control and the protein of interest must be in a linear range.

The final consideration for selecting a loading control is to consider the linear range of the signals. If the signal detected for both the loading control and the protein of interest are not in a linear range, the bands will be over-exposed, or “burnt out,” and you’ll be unable to perform quantitative analysis. 

Related Blog: Am I setting up my loading controls correctly?

The fix? In this case, prevention is the best medicine. It may be necessary to run a titration of your experimental samples to determine the dynamic range of the protein of interest and the loading control. Some care in advance can prevent having to go back and repeat those blots, just to get quantitative data. We recommend loading 20–50 μg of total protein per lane on a mini gel. For most target proteins and model systems, this is the optimal total protein range when probing with CST antibodies.

Loading Control Antibody Sampler Kits from CST

CST has two loading control antibody sampler kits that can help you determine the ideal control for your experiment. For example, the Loading Control Antibody Sampler Kit (Rabbit) #5142 includes antibodies for the following common loading controls:

• β-actin: Actin is a ubiquitous protein and a major component of the eukaryotic cytoskeleton. Actin exists mainly as the F-actin fibrous polymer.
• Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): GAPDH catalyzes the phosphorylation of glyceraldehyde-3-phosphate during glycolysis. Recent work has demonstrated that GAPDH plays roles in apoptosis, gene expression, and nuclear transport.
• Histone H3: Histone proteins, including histone H3, make up the primary building block of chromatin known as nucleosomes. Modulation of the chromatin structure plays an important role in the regulation of transcription in eukaryotes.
• β-tubulin: Globular tubulin subunits made up of α- and β-tubulin heterodimers are the building blocks of microtubules, one of three types of cytosolic fibers that comprise the cytoskeleton.
• COX IV: Cytochrome c oxidase (COX) is a hetero-oligomeric enzyme consisting of 13 subunits localized to the inner mitochondrial membrane. It is the terminal enzyme complex in the respiratory chain, catalyzing the reduction of protons across the mitochondrial inner membrane to drive ATP synthesis.

A similar antibody sampler kit produced in mice, #9774, is also available.

Additional Resources: 

• Visit our Western Blot (WB) Resource Center for all things WB.