25th July, 2017

AptaBlot – increasing the utility of Western Blotting

Western Blotting is a powerful technique used to detect and identify proteins. Antibodies have traditionally been used as the detection molecule, but aptamers are now emerging to complement or address shortfalls of existing reagents.

Basis of the Western Blot

The first step of a Western blot uses electrophoresis to separate proteins in a gel. Samples are then transferred to a nitrocellulose membrane and blocked to reduce nonspecific binding. The membrane is then exposed to a primary antibody specific for the target of interest. Detection occurs in two ways; either the primary antibody is directly conjugated to a reporter molecule, or a secondary antibody is used specific to the primary. Reporter molecules include fluorescent tags, radiolabels and enzymes that react with chemiluminescent or colourimetric substrates.

The advantage of secondary antibodies is the ability to use them alongside multiple primary antibodies. For example, if the primary antibody is produced in a mouse, the secondary antibody will be anti-mouse.

Adapting the Western Blot for AptaBlot

The success of a Western blot is dictated by the specificity of the primary antibody. AptaBlot adapts traditional Western blotting by using aptamers, nucleic acid based affinity molecules. An aptamer can be modified to fit your method of detection by incorporating various reporter molecules or tags, including those recognised by existing secondary antibodies (Figure 1). Aptamers are selected with the necessary modifications in mind, ensuring they are fit-for-purpose and easily integrated with current technology.

 

The actions for possible aptamer based Western Blotting.

Figure 1:
A) 1 step Western Blot using an aptamer as both the affinity reagent and reporter molecule.
B) Classical 2 step Western Blot using an aptamer as the primary binding molecule and a secondary antibody as the reporter.

One of the limitations of using antibodies is the restricted number of targets they can interact with. Antibody generation requires the target to be an antigen eliciting an immune response. In contrast, aptamer selection can occur with non-immunogenic targets. Aptamers can bind to proteins, small molecules, other large molecules (eg. carbohydrates), viruses, cells and whole tissues. In vitro selection of aptamers means they can be generated against toxic compounds, which is not possible with antibody technology.

Aptamers can also be generated rapidly. Antibody production can take up to 12 months, aptamers can be selected to a target in 12 weeks. Coupled with limited batch-to-batch variability during synthesis, aptamers are a powerful addition to existing Western blotting methods.

If you want to find out more about AptaBlot or any other aptamer technology, please get in touch using the form below.

Aptamer Group – Delivering on Target