Product Survey: Western Blotting Transfer Systems

Changing of the Guard
by Harald Zähringer, Labtimes 04/2016

Protein transfer in Western blotting is still performed in many labs in the traditional way with tank or semi-dry blotters. But alternative Western blotting systems based on capillary electrophoresis or microfluidic chips are gradually gaining ground.

There is hardly another life science technique that has changed so little over time than Western blotting. Established independently in the late seventies by Neil Burnette, George Stark (both from the US west coast, hence the wordplay) and Harry Towbin from Switzerland in slightly different implementations, the classical Western Blot is still one of the most employed life science methods – regardless of its many shortcomings and pitfalls.

Simple but effective

One reason for the prolonged success story is the simple setting of a Western. In its most basic form, introduced by George Stark’s group, the protein transfer from the electrophoresis gel to the blot membrane even runs without a power supply by simply utilising the capillary forces of a stack of filter papers: just place the membrane onto the gel, add a few filter papers soaked in transfer buffer, put a weight on top of the stack and give the proteins enough time to migrate out of the gel onto the surface of the membrane – very old-school but still in use. And not the worst way to accomplish a perfect blot that represents the complete spectrum of separated proteins, including very low as well as very high molecular weight proteins.

Capillary Westerns may be performed with simple standard equipment available in every ordinary lab, such as plastic trays, plexiglass plates and some kind of weight. Commercial capillary blotters, which are still offered by specialised companies, usually apply a bracket (pushed down with screws) that depresses the cover plate placed on top of the blotting staple. This assures a tight contact between blotting paper, membrane and gel and prevents the formation of air bubbles, which may interfere with protein transfer. But even this improved set-up cannot zero out the major drawback of capillary Western blotting: the protein transfer is awfully slow. It usually takes several hours or overnight to complete and requires a lot of time and patience – which most researchers don’t have.

Hence, Harry Towbin came up with the idea to install the blot sandwich made of gel, nitrocellulose membrane and filter papers in a gel destainer, filled with either diluted acetic acid for blotting of urea gels or the classical Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol) for blotting of SDS-PAGE gels. He connected the anode and cathode of the destainer to the respective pins of a power supply, and adjusted voltage and current to levels that enabled fast electrophoretic migration of the proteins without too much heat production.

Something went wrong with this Westen blot but who’s to blame? Outdated transfer buffer, degraded proteins, too much heat development or just bad luck? Photo: Azaz Khan

Towbin’s so-called tank or wet blot technique hasn’t changed much over the years. Granted, tank blots are no longer performed in destainers. Current manufacturers of blotting equipment offer stylish blot apparatuses in different sizes, usually provided with closely-spaced wire electrodes, to provide strong electrical fields. Researchers have also developed new blotting buffer systems, such as CAPS buffers, however; Towbin’s original recipe is still widely used in many labs as a standard buffer.

Speeding up protein transfer

Tank blotting is faster than capillary blotting but it still takes about one to three hours until the protein transfer is finished. Transfer speed may be enhanced with semi-dry blotting systems, equipped with inert graphite or carbon plate electrodes on the bottom and the top of the blot apparatus. The semi-dry blot sandwich consists of a pre-wetted extra thick filter paper, membrane, gel and a second pre-wetted filter paper – all exactly cut to the size of the electrodes. After closing the lid, the sandwich is tightly squeezed between the two electrodes, allowing maximum current to flow through the gel, leading to a strong electrical field that cuts down transfer time to about 30 minutes to one hour.

Reducing the amount of buffer even further gave rise to dry blotters with extremely short transfer times of only a few minutes. Dry blotters utilise gel matrices functioning as ion reservoirs, instead of wetted filter papers as well as copper electrodes in place of inert graphite anodes and cathodes. This modified design has two advantages: in contrast to graphite electrodes (which electrolyse water), copper electrodes do not generate oxygen bubbles that may disturb protein transfer; the minimised distance between the copper electrodes allows high field strength, enabling very fast transfer.

Smart researchers combine dry blotting with the fast Bis-Tris gel electrophoresis system, to execute the first two steps of the Western blotting process in less than 45 minutes. According to a recent paper by Jillian Silva and Martin McMahon from the University of California, that’s the “fastest Western in town” – the fastest classic Western to be precise (J Vis Exp, 84 e51149). But besides the fact that 45 minutes, plus the additional time needed for protein detection and washing steps, is not really “fast”, traditional Western blotting has some more severe drawbacks: it is still very labour-intensive and requires a lot of manual work, it swallows up tons of proteins per assay and last but not least, it is only poorly suitable for multiplexing, i.e. multi-protein analysis.

Hence, researchers have developed several non-traditional Western blotting concepts, mostly based on capillary electrophoresis (CE) or capillary gel electrophoresis (CGE) techniques and microfluidic systems. Capillary electrophoresis was already introduced back in the early nineteen eighties but has been applied in Western blotting devices only recently. There are basically two major approaches of capillary Western blots. One is based on a technique developed by Robert Kennedy’s group at the University of Michigan, the other has been put forward by Roger O’Neill and his co-workers from the US company Cell Biosciences, now ProteinSimple.

Kennedy came up with the idea to separate the protein mixture in a gel-filled capillary, surrounded at the end by a sheet capillary, which has direct contact to a blotting membrane. The blot membrane is moved along the x-axis by a translational stage, to capture proteins exiting the capillary on the membrane. Detection of the deposited proteins is done similarly to a classic Western with an immunoassay; the complete process takes about one hour.

Western without blotting

O’Neill’s group went one step further by skipping the blotting step altogether. Similar to the approach of the Kennedy group, proteins are separated in the first step by capillary electrophoresis. However, O’Neill and his co-workers utilised a capillary filled with a sieving media (capillary gel electrophoresis) and immobilised the separated proteins onto the inner surface of the capillary with a special capture chemistry. Primary and secondary antibodies are then flushed through the capillary to detect target proteins inside the capillary, eliminating the blotting step of traditional Westerns. ProteinSimple’s so called “simple Western” system runs automatically and requires only small volumes (40 nL) of sample material.

Microfluidic Western blotting approaches try to integrate the western workflow in microfluidic chips, containing a set of micro-channels usually fabricated from glass, silicon or polymers, such as Polydimethylsiloxan (PDMS). A very interesting microfluidic device suitable for multiplexed Western blotting has been published by the Kennedy group in the June edition of Analytical Chemistry (Shi Jin et al., 88, 6703-10). The blot device is basically a miniaturisation of Kennedy’s CE-blotting system described above. Instead in a capillary, however, Shin et al. separated the proteins in the tiny channel of a glass chip filled with a sieving media. Similarly to the CE-Western, the microfluidic chip is dragged along a PVDF blotting membrane to deposit the proteins flowing out of the channel’s exit onto the surface of the membrane. The trick to obtain multiplexed Western blots is pretty simple: the same protein sample is repeatedly injected into the chip, leading to multiple protein tracks on the membrane that can be probed with different antibodies. According to the authors, as little as 400 ng of total protein, injected into nine fractions, were sufficient to detect eleven different proteins.

Single cell Western

Another notable microchip-based, western blot device has been put forward by Amy Herr’s group at the University of California, Berkeley. The group developed a single cell Western blot assay that enables parallel analysis of thousands of individual mammalian cells (Nature Methods, 2014, 11, 749-55).

The basic idea of Herr’s single cell Western blot is pretty straightforward. Single cells are captured in the tiny microwells of a 30 µm thick, photoactivatable ­polyacrylamide gel (PACT-gel), coated onto a silicon wafer (with dimensions of a microscope slide). After a washing step, the individual cells in the microwells are lysed within a few seconds by simply pouring a lysis buffer onto the chip.

An electrical field is applied to the chip that drives the proteins of the lysed cells into the PACT-gel layer, where they are separated according to their size. After 45 seconds a UV light is switched on that triggers the crosslinking (blotting) of proteins to the gel. Lysis, electrophoresis and “blotting” took a mere 75 seconds: Herr’s single cell western transfer technique is not only super clever, it is undisputably the fastest in town.

But there’s a fly in the ointment. The in-gel immunoprobing of single cell Westerns via primary and secondary fluorescent ­labelled antibodies as well as the

First published in Labtimes 04/2016. We give no guarantee and assume no liability for article and PDF-download.

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