Western blotting is a biochemical technique used to identify specific proteins in a complex sample mixture. It combines electrophoretic sieving with immunoassay to provide semiquantitative assays of proteins. It was first described in 1979 ^{[1, 2]} and has become a workhorse in biochemical research. This method is routinely implemented for basic research and as a confirmative test for clinical assays and regulatory tests because of high selectivity conferred by using both separation and immunoassay. In biomedicine,Western blot is used as a diagnostic tool for several diseases including HIV,Lyme disease,and Hepatitis B ^{[3, 4]}. In a Western blot,proteins are separated by size using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a membrane by electroblotting. The membrane is then treated with blocking solution and then probed sequentially with primary and secondary antibody to detect target protein.

Although reliable,robust,and widely adapted,Western blotting has some well-known drawbacks. It is time consuming,usually requiring 8-20 h to complete including gel preparation,sample treatment,separation,transfer,multiple incubations,and washings. Most of these steps are performed manually which makes them labor intensive and can decrease reproducibility and quantification. The Western blot has not been miniaturized,which wastes materials and reduces sensitivity. Usually 10-15 μL sample is needed for one assay,which is not suitable for sample limited applications. A related problem is the difficulty of determining multiple proteins in one sample. Multi-protein analysis is accomplished by removing antibody from the blotting membrane (also called ‘‘stripping’’) followed by reprobing with antibodies for the next protein target. This requires re-performing the slowest step of the process. Analysis of large proteins is hindered by difficulty of transferring them from slab gels onto membranes. Recently several improvements in the Western blot process have been reported. These improvements include development of microscale counterparts and platforms which enable rapid and reliable Western blot type assays. Several of these technologies have been commercialized. 2. Western blotting using capillary electrophoresis

A potentialway to improveWestern blotting is to adapt capillary electrophoresis (CE) for the separation. CE is a powerful separation technique,which allows for rapid separation with small sample requirements. A variety of CE separations have been developed including free solution capillary zone electrophoresis (CZE),capillary isoelectric focusing (cIEF),capillary gel electrophoresis (CGE), and micellarelectrokinetic capillary chromatography (MEKC). In CZE,analytes migrate according to their electrophoretic mobility. CGE allow sieving separations. The gel media may be a crosslinked polyacrylamide gel or entangled polymer solution. In both cases, electroosmotic flowis suppressed due to the surface electric double layer suppression. As is well known,the ratio of SDS-protein for most globular proteins is 1.4mg SDS per gramof protein yielding an approximately constant electrophoretic mobility for all proteins because the amount of negative charge added is dependent on protein size ^[5]. Because of the uniformcharge to size ratioamong all the unfold peptide chains,the intrinsic amino acid sequence does not affect the migration time. In a sieving media,only the length of the polypeptide chain,i.e. the size of the protein controls the migration rate. Thus,SDS-protein complexes are separated by size in CGE. Compared to SDS-PAGE on a slab gel platform,capillary gel electrophoresis enables sample size reduction,automation,repeatable separations of one sample,and faster analysis time due to compatibility with higher electric field. The CGEmethod also allows rapid estimates of molecular weight ^[6].

Despite the long history of CGE,only recently was this technique coupled with blotting to enable a CE-based Western blot ^[7]. This was a hybrid approach in which the exit of sieving electrophoresis capillary was interfaced to a blotting membrane so that separated proteins were captured on a moving PVDF membrane as they exit the column. The time of analysis is reduced through faster separation (40 min for up to 155 kDa proteins), owing to the higher electric field possible on CGE compared to conventional slab gels,and elimination of the electro-blotting step. Using a fast immunoassay method,a full Western blot for lysozyme was completed in 1 h with 50 pg mass limit of detection (LOD). The lowmass LODwas due to the lowvolume injection and confinement of the separated zones to a small spot. The system has potential for improvements in speed,throughput,and sensitivity. An interesting possibility is that because the proteins are captured on a membrane,they are accessible for or other chemical tests besides the immunodetection. For example, capillary electrophoresis and matrix-assisted laser desorption/ ionization mass spectrometry (MALDI/MS) have been combined to provide separation and mass analysis of peptide and protein mixtures in the attomole range using similar membrane capture ^{[8, 9]}. Thus,this system allowed a faster Western blot with lower sample consumption,elimination of a separate blotting step,and increased automation. Another advantage was the use of entangled polymer solution as the sieving media. This media can be readily replaced by pumping it out; in contrast to crosslinked polymers which have limited lifetime and require extensive regeneration procedures. Nevertheless,this method was hindered by the need to have a post-column flow,requiring some careful engineering.

An exciting form of a CE-Western blot was reported by O’Neill and coworkers ^[10]. High-resolution capillary isoelectric focusing (cIEF) was used to separate proteins in a 400 nL capillary. The separated proteins were then immobilized on the pretreated capillary wall surfaces by photoactivated cross-linking. The captured target proteins can be probed by flowing primary antibodies through the capillary. This high-resolution cIEF is especially useful for separating protein isoforms and modifications, e.g. phosphorylation,of proteins. This method combined with rapid photochemical capture method has low sample consumption and the potential for automation. A potential drawback is that the photoactivated capturing efficiency, ~0.01% ^[11] limits the sensitivity of this method. A fully automated form has been commercialized by ProteinSimple. 3. Western blotting using microfluidic chips

Another approach to improve Western blot assays is through the use of microfabricated fluidic systems. Microfluidics is a platform to manipulate fluids in channels with dimensions of tens of micrometers. Advantages of using microfluidic platforms for analysis include short analysis time,relatively low cost,low sample consumption,and potential for multiplexing and easy automation. These advantages were convincingly demonstrated in the initial report on using a microfluidic chip for protein sieving electrophoresis. In this study,high-speed separation (1 min over 1.25 cm long channel) and high separation efficiency (10⁷ plates/m or 3750 plates/s) were achieved for a protein size ladder ^[12]. A key factor in achieve such excellent separation results was injection of a well-defined sample plug (about 25 pL) using the so-called pinched injection method. Despite this early success with protein size separations by microfluidics ^[13], it was many years for a microfluidic Western was demonstrated.

In this regard,an elegant approach was that described by Herr’s group who developed integrated microfluidic systems that enable protein sieving on a chip and then directing toward an antibody capture region on-chip ^{[14, 15, 16, 17]}. These systems have demonstrated Western blot results in a rapid,miniaturized format. Upon separation completion in a precast PAGE region of the 1mm× 1.5 mm chamber,proteins are driven by electrophoretic force toward antibody-patterned polyacrylamide gel blotting region for immunoblotting. By adding more antibody capturing zones next to the separation region,multianalyte on-chip Western is achieved. These methods are fast and sensitive,but do require covalent antibody immobilization on the chip. Another approach developed by the same group uses photoactivated cross-linking method to immobilize proteins on gel permanently through covalent bonds after size-based separation ^{[11, 18]}. The key to this approach is the new functionalities of a light-responsive material, allowing the switch from photo-polymerization to photo-immobilization. Immunoassay is then performed by flowing primary and secondary antibodies through the separation region,followed by conventional imaging. Analyses of human sera for HIV immunoreactivity and cell lysates were used to validate the microfluidic assay. The 1-inch by 3-inch compact platform supports 48 micro Western blots within 60 min. Also,by cross-linking to the gel, capture efficiency is enhanced relative to cross-linking on the wall as described above.

In another approach,Jin et al. extended the approach of using hybrid CE-protein capturing membrane apparatus by using a microfabricated glass chip for protein sieving separation ^[19]. Relative to the CE system,the use of a microfluidic system greatly improves the speed of separation,automates injection, and improves reliability and performance of interface to membrane. The use of microfabricated sheath-flow greatly stabilizes current and reduces band broadening as proteins migrate from separation channel to the membrane. An attribute of this system was that different samples could be deposited in different regions of a membrane enabling multiple samples or proteins to be evaluated. To evaluate the utility of this system, actin and AMP-activated protein kinase in INS-1 cell lysate and lysozyme in egg whites were detected. Detection limits are 0.7 nmol/L for lysozyme and 2.4 nmol/L for actin,which compare well to other microfluidic Western blots ^[15]. The technology also enables more than 50 replicate separations from a single sample thus greatly increasing the number of potential probings.

Another use of microfluidics has been for application of reagents to a membrane from conventional slab gel PAGE ^[20]. In this method,once a separation is done and transferred from the gel to a membrane,a fabricated microfluidic network with 5 microchannels is placed over the protein bands and sealed to the membrane. Multiple proteins are simultaneously probed by flowing different antibodies through the 5 channels. Comparing the antibody consumption with conventional Western blot protocols,the new antibody introduction approach significantly reduces the volume needed to less than 1 μL in each microchannel.

Several commercial products have been released to address different stages of Western blotting. This is an ever-growing field with an estimated $1 billion global market ^{[22, 23]} as the technique remains popular. Agilent developed a high-speed microfluidic gel electrophoresis protein separation platform (Agilent 2100 Bioanalyzer) ^[24]. The instrument automates conditioning,sample loading,separation and detection,though there is no immunoaffinity steps to this instrument.

Millipore has released a second version of its fast immunoassay module,called SNAP i.d. 2.0 protein detection system ^[25]. The instrument enables rapid blocking,antibody incubation,and washing by applying vacuum to pull solutions through the blots after separation and transfer are done. By shortening the time for immunoassay from 8 to 12 h in traditional protocols to less than 30 min,the SNAP i.d. system offers high throughput analysis and optimization the immunodetection conditions. The system also enables antibody recollection to further reduce the reagents cost.

Life Technologies introduced a fast Western transfer system called iBlot,which features 7 min protein transfer from gel to membrane ^[26]. The iBlot dry blotting requires no transfer buffer and no external power supplies. The rapid transfer is possibly due to the unique design to reduce the distance between the electrodes and the integrated power supply so that the system could generate a high field and increase transfer speed. Another product released by the same company recently is called iBind,which compresses overnight incubation time to about 2.5 h. The instrument requires no electricity or battery to drive,automates all antibody incubation and washing steps,and is compatible with most secondary antibodies and detection systems. The sequential lateral flow technology employed in the iBind system allows for timely release of antibodies onto the blots and a constant flow rate over the incubated membrane.

Simple Western developed by ProteinSimple,is a direct outgrowth of the original O’Neill method ^[10] and provides a commercial solution to automate the entire Western blot procedure ^[27]. CGE or cIEF is followed by photoactivated protein crosslinking on capillary walls. Probing is achieved by sequentially flowing antibodies through the capillary,and antibodies bound to the targets can be detected by chemiluminescence or chemifluorescence. 5. Conclusion

Separation methods coupled with affinity interactions are important techniques for life science research. Western blotting, since its first introduction,has become a ubiquitous tool in both basic research as well as clinical diagnosis of certain disease. Recent success in developing miniaturized platforms and integration improve one or more aspects of this powerful but labor intensive and low throughput technique. Looking forward,protein measurement tools that enable high-throughput analysis,high information content,and low costs have great promise for challenging traditional analytical methods and expansion not only in bioscience but also in the clinic.

Our work in this area was supported by NIH grants R37 DK046960,RO1 GM102236 and 1R43GM112289-01.