These tools are making contributions such as elucidating genome diversity and identifying mutations that occur during gametogenesis 1. In particular, RNA sequencing and genotyping on ten to thousands of single cells is possible, owing to microwell arrays and pneumatic microfluidic valves that control RT–PCR 1, 7– 11. The small device length scales and precision fluid control can maintain high local concentrations of single-cell lysates 6. Single-cell measurement techniques capture cell-to-cell variation, and microfluidic tools have a central role in bringing biochemical assays to single-cell resolution. Unfortunately, conventional bulk measurements mask the biology occurring in each individual cell 5. Cell-to-cell variation affects biological systems, from development 1 to stem cell biology 2 to cancer 3, 4. the technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. to extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. We also describe microdevice fabrication for both uniform and pore-gradient microgels. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. scWB involves five main stages: (i) gravity settling of cells into microwells (ii) chemical lysis of cells in each microwell (iii) PAGE of each single-cell lysate (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix and (v) in-gel immunoprobing of immobilized proteins. the gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. ![]() scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state.
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