Including controls in every experiment is essential, and western blot is no exception. Controls help you to recognize unexpected sources of error, both random and systemic, and fix these before they compromise your results.

Positive control lysate

A positive control lysate is a lysate from a cell line or tissue sample known to express the protein you are detecting. A positive result from the positive control, even if the test samples are negative, will indicate that the procedure and reagents are optimized and working. It will, therefore, verify the validity of any negative results. On the contrary, a negative result in the positive control lane suggests that at least one of the steps in your protocol didn't work correctly or there's a problem with your antibody.

We strongly recommend using a positive control lysate when setting up a new experiment; this will give you immediate confidence in the protocol.

Tip: If the expression of your target protein isn't well characterized, use a lysate from cell lines or tissues overexpressing your protein of interest as a positive control. We prefer using cell lysate to tissue lysate as a positive control because protein expression levels in tissue may be easily affected by individual differences or heterogeneity and easily cause lysate batch differences.

For modified targets, like phosphorylation-modified proteins, we suggest using a total antibody as a control.For proteins regulated by drug treatment, we suggest using a control antibody, which exists in the same signaling pathway to validate that your signalling pathway has been activated successfully.

We recommend you check the antibody datasheet, which will often provide a suggested positive control. Always ensure the tissue or cell line you use is from a tested species. Not all the datasheets will have a suggested suitable control, and we recommend the following in these circumstances:

• Check if there are any citations and product reviews for the antibody. Any tissues, cells, or lysates that have been used successfully by these customers can be considered a suitable positive control.
• Check the Swiss-Prot or Omnigene database links on the datasheet. These databases will often list tissues expressing your protein of interest. These can also be considered suitable positive controls.
• Check the GeneCards or Human Protein Atlas entry for the protein. This will usually provide you with relative protein expression levels in various tissues and/or cell lines.
• If you still have difficulty finding a suitable control, we recommend doing a quick literature search on PubMed to see which tissues and cells express the protein of interest.

If you are using online databases, such as Swiss-Prot, Omnigene, or Human Protein Atlas, consider where the data has come from. Thus, the data based on mRNA expression may not correlate well with detectable protein levels.

See our range of lysates hereIf you are using online databases, such as Swiss-Prot, Omnigene, or Human Protein Atlas, consider where the data has come from. Thus, the data based on mRNA expression may not correlate well with detectable protein levels.

Negative control lysate

Negative control lysate is a lysate from a cell line or tissue sample known not to express the protein you are detecting. A negative control will allow you to check for non-specific binding of your antibody and false-positive results.

Tip: Use a lysate from a validated knockout cell line or tissue sample as a negative control.

Loading controls

Loading controls are proteins that exhibit high-level, constitutive expression in the cell type or sample that you are studying. Housekeeping genes, such as actin, GAPDH, tubulin, or mitochondrial proteins, are usually good choices.

Loading controls confirm that protein loading is the same across the gel and, therefore, help to normalize the protein levels you detect. Loading controls also ensure the reliability of your data between samples since the expression levels of the loading control should remain consistent between the different sample types.

Below is an example of negative and loading controls in western blotting (Figure 1).

Figure 1. Western blot with anti-beta actin antibody [AC-15] (ab6276). Lane 1: Wild-type HAP1 cell lysate (20 µg). Lane 2: Beta-actin knockout HAP1 cell lysate (20 µg), used as a negative control. Lanes 1 and 2: Merged signal (red and green). Green - beta-actin (ab6276) observed at 42 kDa. Red - loading control (ab181602) observed at 37 kDa. Ab6276 was shown to specifically react with beta-actin in wild-type HAP1 cells (lane 1). No band was observed in the negative control (beta-actin knockout cell lysate in lane 2).

Here are the main reasons you need to be using loading controls:

• Quantification: when lanes have not been loaded evenly, loading controls can be used to quantify the protein amounts in each lane by using the density of the loading control band to correct for the differences in loading.
• Equal transfer: loading controls have a second role as a control in western blots. They can be used to check that there has been even transfer from the gel to the membrane across the whole gel. This is imperative when comparing the protein expression levels between samples.
• Edge effect: this is an issue that is particularly important in signaling assays or experiments where a large number of lanes are being run at once. Proteins in the outer lanes of the gel are transferred to the membranes in a position close to the frame. This may result in more variation in binding compared to other areas of the gel. Loading controls can show if this effect has occurred and allows us to correct for the variation in binding.
• Requested by referees: using loading controls is essential for publication-quality work. As an example, to be published in many Nature journals, a paper must meet four general criteria, the first of which is that it must "provide strong evidence for its conclusions." This directly correlates to the necessity of controls to prove that the results obtained are valid.

Use the table below to select the right loading control for your sample type. Note it's essential to choose a loading control with a different molecular weight than the protein of interest. This ensures that you will be able to distinguish between the bands.

*Not suitable for samples where the nuclear envelope is removed.

**Tubulin expression may vary according to resistance to antimicrobial and antimiotic drugs (Sangrajang S et al., 1998; Prasad V et al., 2000).

***Not suitable for skeletal muscle samples. Changes in cell-growth conditions and interactions with extracellular matrix components may after actin protein synthesis (Farmer et al., 1983).

†Some physiological factors, such as hypoxia and diabetes, increase GAPDH expression in certain cell types.

‡Not suitable for samples where DNA is removed.

§Many proteins run at the same 16 kDa size as COX IV.

Endogenous control lysate

We recommend including an endogenous control if you test a recombinant protein sample. Folding of the recombinant protein may differ from the endogenous native form and may prevent antibody access to the epitope.

Always ensure the recombinant protein includes the immunogen sequence for the antibody you are using. An endogenous positive control is important to validate the results and indicate how well the reagents (eg antibodies) and protocol are working.

Recombinant protein and overexpression lysate antibody control

We recommend using an anti-Tag antibody to validate recombinant or overexpressed proteins. Additionally, assessing mRNA levels is also encouraged to validate protein overexpression. Be careful when the immunogen is located at the same end as the tag of the protein because the tag may potentially block the epitope recognized by the antibody.

Always ensure the recombinant or overexpressed protein includes the immunogen sequence for the antibody you are using.

No primary antibody control

This is when you add only a secondary antibody. This control can indicate the occurrence of non-specific binding of the secondary antibody. This is a particularly important step when optimizing a multiplexed western blot, as it confirms secondary antibodies do not interfere with each other and cause off-target binding.

Antibody dilution buffer containing no antibody is used instead of the primary antibody solution at this point in the procedure. The secondary antibody is incubated on the sample in the same way as usual.

Alexa Fluor® labeled loading controls for fluorescent western blot

With the advent of fluorescent western blotting, multiple proteins can now be analyzed simultaneously using different fluorophores.This growing technique has been proven to provide improved linearity and increased reproducibility when compared to standard chemiluminescent detection methods in Western blot.

Fluorescent western blotting works optimally in the near-infrared region of the spectrum in order to avoid the chance of membrane autofluorescence within the visible light range.

Therefore, this technique calls for bright and stable near infrared dyes, such as Alexa Fluor® 680 and Alexa Fluor® 790.

Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.