Stain-Free technology as a normalization tool in Western blot analysis
Section snippets
Sample preparation
LCL cells were harvested 24 h after γ-irradiation (6 Gray) and lysed in 1× SDS sample buffer [12]. After removal of cell debris by centrifugation (20.000g, 15 min), the proteins in the supernatant were denatured for 5 min at 95 °C. Protein concentration of the supernatant was measured with the RC DC protein assay (Bio-Rad, USA).
One-dimensional SDS–electrophoresis and Western blotting
LCL samples, together with marker proteins (Precision Plus Protein unstained standards from Bio-Rad) were separated on Criterion TGX Stain-Free “Any kD” gels (Bio-Rad) for 45
Results and discussion
Western blots are commonly employed to determine changes in expression levels of TPs by comparing the relative abundance present in treated samples with that of corresponding control samples. In this context, it is necessary to ensure that observed changes in expression levels are the consequence of cellular events rather than experimental artifacts. As a first step in the Western blotting workflow, it is critical to establish the linear response range of the read-out techniques being employed.
Concluding remarks
Stain-Free technology offers a novel quality control tool for data normalization in Western blotting workflows. The Stain-Free methodology is capable of detecting and correcting for process irregularities as well as, if not better than, HKPs. Stain-Free suggests the added advantage of providing process evaluation checkpoints throughout the Western blotting process. The ability to evaluate progress through the Western blotting process allows researchers to stop prior to antibody incubation if
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