Transfection Quantification Assays
ELISA, WB & qRT-PCR
The Western Blot is a well utilized assay that detects a single protein in a mixture of proteins. It requires obtaining an antibody that is highly specific to the target protein. The Western blot procedure involves three steps. First, proteins are separated by size using gel electrophoresis. The separated bands are then transferred to a nitrocellulose membrane or solid support, followed by incubation with the antibody specific to the target protein. Antibodies that have not bound their target proteins are removed by additional washing steps and the film is developed to determine a semi-quantitative amount and size of protein that bound with the antibodies. Imaging of the membrane following is accomplished either via fluorescence or a colorimetric enzymatic tag such as HRP.
An alternative to the membrane transfer method is an automated and quantitative western blot instrument manufactured by ProteinSimple, but they are very expensive (over $100,000). However, there are companies that provide this western blot service using the ProteinSimple instruments (see westernblotservice.com). The WES system uses extremely low amounts of protein input, has a better range of sensitivity relative to membrane transfer methods and the automation removes person-to-person variability.
ELISAs (Enzyme Linked Immuno-Sorbant Assay) are used to assess the strength of binding of antibodies (Kd measurements), signal transduction, quantitation of secreted proteins and cell viability. Most contract research organizations will have established ELISA protocols that use their own reagents and will be able to quickly adapt their protocols for new protein targets.
ELISAs rely on monoclonal or polyclonal antibodies that bind to the target protein of interest. It is highly recommended to allot time to perform experiments that identify the most specific antibody and optimize the concentrations and reagents associated with the ELISA assay. The output signal relies on a fluorescent, optical, luminescent or radioactive changes. Biology CROs providing ELISA services (see Altogen Labs) will be able to help optimize and obtain data with a low signal-to-noise ratio.
PCR is a basic technique used for creation of millions of copies of sample nucleic acid in a controlled environment. This time-efficient technique bypasses other methods that use bacteria to increase the amount of DNA. It is a versatile technique with several variants that can serve diverse purposes. Basically, a single strand of DNA can be used as the starting template for duplication, and the newly created copy in turn is replicated again and again until one of the key reagents is depleted. Researchers can selectively amplify desired DNA sequences to determine gene expression levels, mutations, INDELs or genomic copy number. This replication strategy helps scientists study all sizes of DNA, from small 20 bp segments up to kilobases in length.
By incorporating the latest in CCD cameras, LED excitation lamps, fluorescent detection probes, automation and low cost of DNA primers, there are now multiple machines on the market driving this technology to be extremely sensitive in detecting less than 100 starting copies of a template. qRT-PCR (quantitative Reverse Transcription-PCR) enables the detection of small changes in gene expression due to siRNA or miRNA transfection.