Western Blotting Using Microfluidics Protocol

Introduction of Western Blotting Using Microfluidics Protocol

The western blot in combination with polyacrylamide gel electrophoresis has been a valuable research tool for life sciences research. It provides qualitative information on predominantly low-abundance proteins in a biological sample. There are, however, multiple steps in the process involving separation of the proteins on a gel matrix, transfer to a membrane, and probing of the membrane with antibodies specific to the protein of interest. Additional time- and resource-intense steps are required to minimize nonspecific binding and enhance the specificity of antibody binding to the protein of interest. Given the recent biological insights enabled through high-throughput proteomic approaches it would be extremely beneficial were an automated, miniaturized, and rapid western blot system available to validate the findings from the high-throughput proteomic approaches and otherwise. Recent advances have led to the incorporation of microfluidics into western blotting approaches. One approach involves a combination of conventional polyacrylamide gel electrophoresis, transfer of separated proteins onto membranes followed by microfluidic based manipulation to facilitate detection of multiple proteins.

Figure 1. Microfluidic subcellular western blotting reports protein localization to the cytoplasmic or nuclear compartment of single cells. (Kevin A. Yamauchi, et al.; 2017)

Materials of Western Blotting Using Microfluidics Protocol

Typhoon Imager: For capture of fluorescent signals generated from secondary antibodies.

ImageQuant TL8.1 software: Used to quantify the fluorescent signals captured by the imager.

The microfluidic template: The template and microchannels were fabricated in-house using soft lithography on a silicon wafer. The template can be made in any fabrication facility that has a "clean" room.

Methods of Western Blotting Using Microfluidics Protocol

Use a mini-gel system to resolve proteins from the biological sample of interest. A polyacrylamide gel made up of 5 % stacking gel and 10 % running gel will work well for low-molecular-weight proteins. The percentage of acrylamide in the running gels can be altered depending on the protein/s of interest. Generally, a higher percent running gel offers better separation of low-molecular-weight proteins.

Perform electrophoresis at room temperature, first at 80 V for 20 min and then at 120 V for 65 min.

Post-electrophoresis, transfer the separated proteins from the gel to a polyvinylidene difluoride membrane (PVDF) at 260 mA for 1 h, for example using Bio-Rad's mini Trans-Blot system.

After transfer incubate the PVDF membrane at 37 °C for 1 h to facilitate protein absorption to the membrane and minimize diffusion in subsequent microfluidic assay phases.

Place the membrane in 0.1 % Ponceau S stain for about 5 min and wash in 5 % acetic acid for 5 min. Repeat the wash two more times. Place the membrane in distilled water and wash twice more, 5 min each time or until background becomes clear (see Note 1). The limit of detection for Ponceau S is about 250 ng of protein.

Place the fabricated microfluidic template (see Note 2 and also Note 7) with the channels perpendicular to the stained protein bands and seal by gently pressing the template against the membrane.

Fill the microchannels with 0.05 % Tween-20 solution prepared in PBS and let stand for about 1 min to dampen the membrane (see Note 3) followed by 5 % BSA prepared in PBS for 20 min to prevent nonspecific binding.

Apply 1–2μL of the primary antibody diluted in PBS (1:20; see Note 4) to the protein of interest and let incubate for 20 min. Use different channels to probe for different proteins using the respective primary antibodies.

Remove the microfluidic template and rinse the membrane in distilled water.

Soak in isopropanol for 30 s, remove, and place in blocking buffer containing 5 % BSA, prepared in PBS, for 20 min.

Wash the membrane in PBS containing 0.05 % Tween-20 twice, 5 min each time with fresh PBS.

Incubate the membrane with fluorescence-tagged secondary antibody (1:1,000) diluted in PBS containing 5 % BSA and 0.05 % Tween-20 for 30 min with mild agitation.

Rinse the membrane in washing buffer (see Note 5) and wash in the same buffer twice, 5 min each time with fresh buffer.

Scan the bands using a fluorescent imager such as a Typhoon Imager (see Note 6) and quantify the signals using software such as ImageQuant TL8.1.

Notes of Western Blotting Using Microfluidics Protocol

Care should be taken not to wash the membrane after Ponceau S staining with excess distilled water to avoid washing away the stain from the protein bands.

The fabricated network template is soft and gentle pressure is enough to seal it to the membrane.

The solutions are introduced into the microchannels as droplets at the microchannel inlet and applying vacuum to the outlet.

Primary antibodies should be diluted in PBS containing 5 % BSA and 0.25 % Tween-20.

Washing buffer used after incubation with secondary antibody contains 0.05 % Tween-20 in PBS.

The recommended setting for the Typhoon Imager are as follows: Laser—488 nm; emission filter—520 nm; PMT—480 V 7; and pixel size—25 μm.

Dimensions of the microfluidic network are as follows: channel height—100μm; channel width—150 μm; channel length—3.5 cm; gap between channels—1,000μm; and number of channels—10.

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Western Blotting Using Microfluidics Protocol

Introduction of Western Blotting Using Microfluidics Protocol

Materials of Western Blotting Using Microfluidics Protocol

Methods of Western Blotting Using Microfluidics Protocol

Notes of Western Blotting Using Microfluidics Protocol

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