Elsevier

Analytical Biochemistry

Volume 433, Issue 2, 15 February 2013, Pages 105-111
Analytical Biochemistry

Stain-Free technology as a normalization tool in Western blot analysis

https://doi.org/10.1016/j.ab.2012.10.010Get rights and content

Abstract

Western blots are used to specifically measure the relative quantities of proteins of interest in complex biological samples. Quantitative measurements can be subject to error due to process inconsistencies such as uneven protein transfer to the membrane. These non-sample-related variations need to be compensated for by an approach known as normalization. Two approaches to data normalization are commonly employed: housekeeping protein (HKP) normalization and total protein normalization (TPN). In this study, we evaluated the performance of Stain-Free technology as a novel TPN tool for Western blotting experiments in comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a representative of the HKP normalization strategy. The target protein (TP) used for this study was MCM7, a DNA licensing replication factor, which was shown previously to be down-regulated by 20% in irradiated lymphoblastoid cell lines (LCLs). We studied the regulation of MCM7 with a multiplex Western blotting approach based on fluorescently labeled secondary antibodies and found that Stain-Free technology appears to be more reliable, more robust, and more sensitive to small effects of protein regulation when compared with HKP normalization with GAPDH. Stain-Free technology offers the additional advantages of providing checkpoints throughout the Western blotting process by allowing rapid visualization of gel separation and protein transfer.

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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