Western Blot – Multimedia

Western blotting is a very useful and widely adopted lab technique, but the traditional procedure can be long and tedious. Researchers can assess only whether a blot image is captured at the end of the long procedure and the quality of western blot data is sometimes challenged due to poor loading controls.

Traditional western blot workflows require an appropriate sample load to detect both target proteins and loading control proteins. The discrepancy between these quantities often leads to oversaturated protein bands, forcing researchers to sometimes run two duplicate gels or titrate down the antibody concentration in order to get quantitative results.

Reliable western blot data can be generated only when the proper sample amount of protein is used. Loading too much protein leads to signal saturation in western blots, yet too little produces weak signals.

Reliable western blot data require detection of the target and loading control proteins in the linear dynamic range. Many published western blot images show saturated protein bands, indicating that they were not measured in the linear dynamic range. Quantitative analysis based on this type of data is not reliable.

The BD™ Cytometric Bead Array (CBA) Cell Signaling Flex Set system lets you achieve high productivity in cell signaling studies by extracting more data from less sample faster than traditional Western blot techniques.

SiRNA designed to target the human VEGF gene promoter revealed a putative candidate for transcriptional gene silencing. However, mutation or deletion of the target site demonstrated that the observed VEGF knockdown was the result of a sequence-specific off-target effect. Bioinformatic and microarray analyses of genes implicated in VEGF transcriptional control followed by knockdown and Western blotting experiments presented one candidate, GRB2, as an unintended target of the VEGF promoter-specifi

The LacZ reporter fusion assay is an excellent method to precisely quantify knockdown efficiency of siRNA-sequences and can be corroborated by Western blot analysis. Efficient production of Lentiviruses and high transduction efficiency (up to 98%) were achieved on lymphoid B- and T-cells as demonstrated by FACS analysis and GFP expression. Successful knockdown effect with specific shRNA was observed in suspension cells three days after lentiviral infection.