Comparison of Automated and Traditional Western Blotting Methods

Abstract

Traditional Western blotting is one of the most used analytical techniques in biological research. However, it can be time-consuming and suffer from a lack of reproducibility. Consequently, devices with different degrees of automation have been developed. These include semi-automated techniques and fully automated devices that replicate all stages downstream of the sample preparation, including sample size separation, immunoblotting, imaging, and analysis. We directly compared traditional Western blotting with two different automated systems, iBind™ Flex, which is a semi-automated system designed to perform the immunoblotting, and JESS Simple Western™, a fully automated and capillary-based system performing all steps downstream of sample preparation and loading, including imaging and image analysis. We found that a fully automated system can save time and importantly offer valuable sensitivity. This is particularly beneficial for limited sample amounts. The downside of automation is the cost of devices and reagents. Nevertheless, automation can be a good option to increase output and facilitate sensitive protein analyses.

Keywords: JESS Simple Western™; SARS-CoV-2; Western blotting; automation; iBind™ Flex; protein analysis.

Figure 1

Lysates containing RBD proteins run by traditional WB. (a) Image of membranes immunoblotted with anti-SARS-CoV-2 RBD (1:2500) or anti-GAPDH (1:1000) Abs using traditional WB. (b) Floating box plots showing the minimum, maximum, and mean of the band volumes for RBD and GAPDH bands for RBD1–4 samples from three independent experiments that were obtained using Image Lab analysis. Lysates (10, 2.5, and 1 µg/lane) were from 293T cells transfected with plasmids expressing different-sized transgenes containing RBD (RBD1–4) or control transgene (CTRL). MW: PageRuler™ Plus Prestained Protein Ladder (Thermo Scientific).

Figure 2

Lysates containing RBD proteins run using iBind™ Flex. (a) Image of membranes immunoblotted with anti-SARS-CoV-2 RBD (1:2500) or anti-GAPDH (1:1000) Abs using iBind™ Flex. (b) Floating box plots showing the minimum, maximum, and mean of the band volumes from RBD and GAPDH bands for RBD1–4 samples from three independent experiments that were obtained using Image Lab analysis. Lysates (10, 2.5, and 1 µg/lane) were from 293T cells transfected with plasmids expressing different-sized transgenes containing the RBD (RBD1–4) or control transgene (CTRL). MW: PageRuler™ Plus Prestained Protein Ladder (Thermo Scientific).

Figure 3

Normalized RBD expression in traditional and iBind™ Flex membranes. RBD (1–4) expression in both traditional WB (a) and iBind™ Flex membranes (b) was normalized to GAPDH expression using band volume obtained from Image Lab analysis. Top bar diagrams represent quantitation from the representative WBs in Figure 1 and Figure 2. Bottom graphs are floating bar representations (minimum, maximum, and mean) of band volume quantitation from three independent experiments.

Figure 4

Assessment of transgene expression by JESS Simple Western™. Different concentrations (100, 50, 25, 12.5 ng/µL) of lysates from 293T cells transfected with vectors expressing RBD containing transgenes of different sizes (RBD1–4) or control transgene (CTRL) were processed in the fully automated JESS Simple Western™ device, probed with the SARS-CoV-2 RBD (1:50) and GAPDH (1:10) Abs. (a) Portrayal of a traditional blot-like image with a lane view of the samples. (b) An electropherogram representation of RBD1, RBD2, and RBD4 (50 ng/µL) samples for both the anti-RBD (solid line) and anti-GAPDH (dashed lines) analysis. Floating bar diagrams (minimum, maximum, and mean) of the peak areas corresponding to RBD (c) and GAPDH (d) protein from three independent experiments. (e) Floating bar diagram (minimum, maximum, and mean; n = 3) of the normalized RBD expression calculated from the peak areas of RBD and GAPDH signals.