Comparison of photoactivatable crosslinkers for in-gel immunoassays

Tan, Kristine Y.; Desai, Surbhi; Raja, Erum; Etienne, Chris; Webb, Brian; Herr, Amy E.

doi:10.1039/d1an01309b

Cited by 4 publications

(4 citation statements)

References 29 publications

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“…Polyacrylamide gels in polydimethylsiloxane devices (72) have been reported for Western blotting applications where the separation channels are short and are generally in line with polyacrylamide wells designed for single cells. Following the size-based sieving separations the proteins can be immobilized in the gel because it has been previously modified with a cross-linking reagent, for example, benzophenone (73). Detection of specific proteins is achieved by transferring antibodies to the gel with an electrophoresis step after which point fluorescent detection is possible.…”

Section: Sieving Separations That Include Capturementioning

confidence: 99%

Gels in Microscale Electrophoresis

Holland

Casto-Boggess

2023

Annual Rev. Anal. Chem.

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Gel matrices are fundamental to electrophoresis analyses of biopolymers in microscale channels. Both capillary gel and microchannel gel electrophoresis systems have produced fundamental advances in the scientific community. These analytical techniques remain as foundational tools in bioanalytical chemistry and are indispensable in the field of biotherapeutics. This review summarizes the current state of gels in microscale channels and provides a brief description of electrophoretic transport in gels. In addition to the discussion of traditional polymers, several nontraditional gels are introduced. Advances in gel matrices highlighted include selective polymers modified to contain added functionality as well as thermally responsive gels formed through self-assembly. This review discusses cutting-edge applications to challenging areas of discovery in DNA, RNA, protein, and glycan analyses. Finally, emerging techniques that result in multifunctional assays for real-time biochemical processing in capillary and three-dimensional channels are identified.

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Section: Sieving Separations That Include Capturementioning

confidence: 99%

Gels in Microscale Electrophoresis

Holland

Casto-Boggess

2023

Annual Rev. Anal. Chem.

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“…Even for fluorescently labeled antibodies, ∼10-fold higher free-solution concentrations are required compared to traditional membrane immunoblotting to achieve sufficient in-gel concentrations . 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.…”

Section: Introductionmentioning

confidence: 99%

“…18 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. 35 Microfluidic Western blotting allows for decreased use of antibodies through deposition of samples via flow 23,24 and inkjet 22 onto moving membranes like PVDF and nitrocellulose. This also allows for reduced sample volumes and greater sensitivity of detection, 21−24 though not yet a single-cell scale.…”

Section: ■ Introductionmentioning

confidence: 99%

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

Alibekova Long,

Benman,

Petrikas

et al. 2023

Anal. Chem.

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While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein content often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity Western blots on single cells (scWesterns) are attractive because they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by the affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address the sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in the limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates, which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 1000 molecules, a 120-fold improvement. This enables us to detect 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies compared to 84.5% of cells using in-gel detection. These results suggest the compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagentsnot previously accessible for in-gel usefor further signal amplification and detection of low-abundance targets.

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“…Even for fluorescently-labeled antibodies, ~10-fold higher free-solution concentrations are required compared to traditional membrane immunoblotting to achieve sufficient in-gel concentrations 18 . 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 25 . Diffusional resistance can be circumvented using out-of-plane electrotransfer of antibodies 26 or chemical modification of gel pore size (including between assay steps) 27,28 .…”

Section: Introductionmentioning

confidence: 99%

Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification

Long

Benman

Petrikas

et al. 2023

Preprint

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While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein contents often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity western blots on single cells (scWesterns) are attractive since they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 1,000 molecules, a 520-fold improvement. This enables us to detect 85% and 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies respectively, compared to 47% of cells using in-gel detection. These results suggest compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagents - not previously accessible for in-gel use - for further signal amplification and detection of low abundance targets.

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Comparison of photoactivatable crosslinkers for in-gel immunoassays

Abstract: Diazirine-functionalized hydrogels have lower fluorescence background signal compared to benzophenone-functionalized hydrogels for in-gel immunoassays and can improve the lower limit of detection of fluorescence immunoassays.

Cited by 4 publications

References 29 publications

Gels in Microscale Electrophoresis

Gels in Microscale Electrophoresis

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

Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification

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