Perhaps no set of reagents is more critical in the technique of Western blotting than the antibodies.
Antibodies selectively bind their target protein, which has been separated by gel electrophoresis and transferred to a blotting membrane.
Because the signal generated by the binding is proportional to the amount of protein in the sample, the user can determine the level of gene expression or the presence of any given protein or biomarker.
Without the right antibody to properly detect the target antigen, there can be no satisfactory experimental result.
That is why early in the Western blot workflow, it is important to determine the antibodies that will best fit the experiment.
Primary and secondary antibodies for Western blot
Western blotting generally relies on a pair of antibodies, the primary and secondary antibodies.
The primary antibody, which is applied first, aims to recognize the target protein, specifically and selectively binding to it.
The secondary antibody’s purpose is to then selectively bind the primary antibody and produce a reliable signal for detection, generally via a conjugated enzyme label like HRP or AP.
Secondary antibodies are specific to the host species of the primary antibody.
Multiple secondary antibodies can also bind a single primary antibody, effectively amplifying the detectable signal for the blot.
A conjugated primary antibody may also be used to perform a direct Western blot, circumventing the need for a secondary antibody.
However, this loses the benefit of signal amplification.
Considerations for choosing antibodies for Western blot
Confirm the specificity of the antibody.
Antibody specificity is the ability to recognize and bind the target antigen, largely determined by the immunogen the antibody was raised against.
Verification of the immunogen can be as simple as referring to the supplier’s antibody product page.
Determine if the protein or peptide sequence used to immunize an animal correctly matches the intended protein of interest of the correct species.
This is good practice, as sometimes the product may use an alias name of the protein that is also shared by a different protein.
When running native western blots, it is ideal to choose primary antibodies that are raised against the purified intact protein as they may not recognize their binding epitopes when presented in a linearized form.
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Determine the selectivity of the antibody.
Antibody selectivity, or its cross-reactivity, is the preference to bind its target antigen in the presence of other competing proteins within a complex mixture.
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An antibody with a high degree of non-selectivity can potentially bind unintended targets, leading to misleading bands within the blot.
Cross-reactivity may also occur with proteins from other species aside from the intended host species of the primary antibody.
As a general rule, primary antibodies should come from a different species as the target protein to further minimize the secondary antibody binding with other proteins coming from the sample.
To account for more potential issues with selectivity, refer to the supplier’s antibody product page for cross-reactivity information.
Check for antibody validation data for Western blotting.
Antibody performance, along with specificity and selectivity, can vary depending on the type of assay or application.
In this case, it is ideal to use antibodies validated for specific use in Western blotting.
Validation is the experimental proof and documentation that a specific antibody is suitable for an intended application or purpose.
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Suppliers often test their antibody products and document validated applications as well as other characteristics in the antibody datasheet.
Users should also look for the tested sample type, any application-specific conditions, and testing methods.
Thorough validation testing can include methods such as gene knockdowns, lot-to-lot testing, and multi-cell line and species analyses.
Independent sources can also provide insight into antibody quality.
These can include scientific publications and online resources that use published Western blot data.
In particular, many antibodies mentioned in the Human Protein Atlas have been validated for Western blot, following a proposed standard set of ‘validation pillars.’
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Get more insights from the research community.
As Western blot is a very widely used application, there is some likelihood that another researcher has previously used an antibody on a similar target protein.
A published figure using a similar sample type or experimental condition may lead to finding an ideal product, or—inversely—rule out a potentially problematic antibody.
The Biocompare Antibody Search Tool lets users search for products with citations, figures, or reviews.
See if your protein of interest has been used by others for Western blotting.
Choose the right control antibodies.
Every experiment needs proper controls and Western blots are no different.
Examples of commonly used immunoblotting loading controls are antibodies for housekeeping genes such as GAPDH, beta actin, beta tubulin.
However, users should be aware that certain conditions can affect the detection of some housekeeping genes.
Their expression levels can vary under certain conditions, such as hypoxia, serum starvation, exercise, transplantation, cell culture density, and even tissue variation.
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Under these conditions it would be prudent to review the literature for the validity of using these controls.