Western Blots

Western Blots / Western blotting is a well 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
  • Less 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 compare to animal facilities

Dot Blots

Whilst less common that western blotting, dot blots allows 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 allows the researcher to perform experiments in a high-throughput, cost effective manner.

 

Representative dot blots

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.