Capillary electrophoresis Western blot using inkjet transfer to membrane

Enhancing Single-Cell Western Blotting Sensitivity Using Diffusive Analyte Blotting and Antibody Conjugate Amplification

“…Diffusive transfer of protein species from polyacrylamide gels to blotting media has been reported for traditional separation assays. − However, its performance has not been validated at spatial scales relevant to scWesterns. We reasoned that transferring protein species from single-cell separations to a nitrocellulose paper interface − ,,− would increase sensitivity by mitigating subsequent mass transfer limitations during probing. We reasoned that nitrocellulose was a superior choice for several reasons.…”

“…Two main groups of methods are currently employed in single-cell proteomics, including highly sensitive antibody-based methods and highly multiplexed mass spectrometry. − Among antibody-based methods, microfluidic assays that run on open-faced hydrogels have significant advantages in simplicity and modularity, enabling several analytical advances. − Hydrogel matrices can sieve, concentrate, or immobilize proteins, enabling the quantitation of species in downstream assay steps. Further, detection of microscale antigen distributions in microfluidic devices can benefit from “ambient assay kinetics” as measurements are independent of the sample volume and do not change analyte concentration, thus allowing for a lower limit of detection. − However, hydrogel properties favorable for one step are often not favorable for others and can create conflicts of performance between them.…”

“…This in turn can result in relatively high background staining of gels due to physical trapping of unbound antibody, limiting assay performance and increasing cost . Microfluidic Western blotting allows for decreased use of antibodies through deposition of samples via flow , and inkjet onto moving membranes like PVDF and nitrocellulose. This also allows for reduced sample volumes and greater sensitivity of detection, − though not yet a single-cell scale.…”

“…Microfluidic Western blotting allows for decreased use of antibodies through deposition of samples via flow , and inkjet onto moving membranes like PVDF and nitrocellulose. This also allows for reduced sample volumes and greater sensitivity of detection, − though not yet a single-cell scale. Diffusional resistance can be circumvented using out-of-plane electrotransfer of antibodies to significantly improve antibody delivery and increase probing sensitivity .…”

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…In addition, reviews by Grochocki [372] (focusing on stacking toward metabolomics), John [373] (preconcentration in nonaqueous CE), and Suntornsuk [374] (preconcentration of pharmaceutical and related substances) have also been recently published. As expected, most of the recent reports involving stacking are focused on the application of the approach to various samples, including drinking water [375, 376], brain tissue [377], fish [378], meat [379], plants [380], human urine and serum [381], noting that the injection of these samples often requires homogenization, filtration, evaporation, and/or dilution [382, 383]. It is also worth mentioning a recent approach by Perrin's group, describing the possibility to combine desalting, protein precipitation, automated liquid–liquid extraction, in‐line CE stacking and electrophoretic separation [384], the possibility to combine surfactants and pressure to increase the concentration of analytes (×3000) [385], and the approach described by Graf coupling ITP–CE for the analysis of glyphosate at pM levels [386].…”