Transfection Quantification Assays (ELISA, WB, qRT-PCR)

ELISA, WB & qRT-PCR

Western Blot

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 cost factors can limit the potential use of such an instrument for single experiments.  However, several companies can offer quantitative blot services for individual experiments. The WES system uses extremely low amounts of protein input, has a better range of sensitivity relative to membrane transfer methods, and removes person-to-person variability.

ELISA

ELISAs (Enzyme Linked Immuno-Sorbent Assays) 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 fluorescent, optical, luminescent, or radioactive changes.  Biology CROs providing ELISA services (see Altogen Labs) can help optimize and obtain data with a low signal-to-noise ratio.

qRT-PCR

PCR is a basic technique used for the 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. PCR begins with a single strand of DNA, which is 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 a 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 technology, and DNA primers, novel machines have driven PCR 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.

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