Western Blots (Western blotting) is an established technique for the analysis of intracellular protein levels. Since blotting relies heavily on antibodies, western blotting is often a frustration for many researchers with poor signal-to-noise ratios and poor target specificity. In addition, most western blotting is currently performed using two antibodies – one recognising the target epitope and a second able to bind to the primary antibody which is linked to a reporter molecule e.g. HRP, fluorophores.
Aptamers offer researchers multiple benefits over antibody based systems:
- Cleaner bands – ability to increase number of fluorophores to increase signal-to-noise ratios
- Fewer non-specific bands – western blotting can be considered the primary outcome during the selection stage, plus the use of counter-selection targets to increase specificity
- Direct conjugation to reporter – no secondary antibody incubation/washing times
- Nucleic acids poorly bind PVDF/nitrocellulose membranes – no membrane blocking steps required
- Lower costs – once developed, aptamers can be provided at a much lower cost than antibodies due to chemical synthesis compared to in-vivo production.
Whilst less common than western blotting, dot blots enables the researcher to immobilise protein material directly to a membrane, without the time consuming process of SDS-PAGE.
The speed of membrane preparation, coupled with the advantages of aptamer technology enables the researcher to perform experiments in a high-throughput, cost effective manner.
Example data of dot blots comparing two polyclonal aptamer pools. A protein of interest was pipetted onto a nitrocellulose membrane and allowed to adhere. Membranes were incubated with either the native library or aptamer pool (1nM) overnight and washed the following day before imaging.