Quantitative Western Blot Analysis
The reproducibility of Western blot analysis along with other immunoasssys is always a concern due to the complex series of steps involved.
Generating accurate quantitative data from WB requires a high level of quality control and is also dependent on user expertise.
It cannot be overstated the importance of correct protein extraction method, proper electrophoresis, transfer of proteins, immunodetection of blotted protein, and the imaging and analyzing of the data. The goal is having the ability to reproduce results on a consistent basis.
Understanding of the biological characteristics of the target protein will allow for optimal protein extraction results from various sample source.
To ensure reproducibility, there needs to be critical validation steps incorporated into the process to minimize errors and variability; this also includes validation of antibodies by both the supplier and end users. It should be noted that the quality of data generated form a WB is correlated to the quality of the sample and reagents used; this is dependent on the ability to prepare quality protein extracts. Another factor to take into account is the relationship between sample loading and band intensity. It is important to identify the linear range of detection; the range of sample loading where the band intensity increases in proportion to sample loading. The next step in the WB sequence is blot processing which is tedious and time consuming due to the number of steps that involve reagent changes; blocking, primary antibody, secondary antibody and washing steps. Since each step is timed, it is virtually impossible to repeat each timed step without automation. Reproducibility can be achieved by using a robotic workstation that automates the blot processing steps from start to finish. Researchers have shown documented improvement in reproducibility, sensitivity, and lower background using Precision Biosystems BlotCycler Touch and Mini. For more information visit us at Blotcycler.com
Need a quotation for the BlotCycler? contact us at info@precisionbiosystems.com
