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Almost all cells studied in vitro and in vivo, including stem cells and cancer cells, do not represent a homogenous population but are inherently heterogeneous at a molecular level.
This determines cellular identity, location, and function within a tissue, organ, and ultimately an organism. Advances in single-cell analysis techniques are making it possible to study the transcriptional profile and protein expression of a single cell within this heterogeneous population.
Single-cell proteomics have thus far been dependent on immunoassays like immunocytochemistry and flow cytometry, which rely heavily on antibody specificity and proper controls. As a result, these techniques can suffer from misinterpretation.
The western blot immunoassay avoids these issues by separating proteins based on molecular weight. Yet, the output of traditional western blot represents an average of all cells within the tested sample, and single-cell analysis is not possible.
The single cell western blotting technique
A team of researchers led by Amy Herr at UC Berkeley has recently demonstrated single-cell quantitative analysis of proteins resolved by molecular weight (Hughes et al., 2014).
Using single-cell western blots (scWesterns) researchers performed multiplexed western blots on up to 2000 individual cells in less than four hours. The technique relies on a thin layer (30 µm) of polyacrylamide gel coated on a glass microscope slide.
The gel is pocketed with thousands of microwells which can capture individual cells. Cells are lysed in microwells after which current is applied and electrophoresis separates single cell protein lysates through the wells into the gel.
Proteins are then cross linked and immobilized in the gel. The thin layer of polyacrylamide allows the slide to be probed with primary antibodies which diffuse into the gel and bind to target proteins.
Similarly, fluorescently labeled secondary antibodies along with a fluorescence microarray scanner are used to visualize the targets.
Slides can be stripped up to 9 rounds with 50% signal recovery owing to the robust photoactive protein immobilization technique. This makes single cell westerns suitable for multiplexing, archiving and reusing single cell lysates.
Abcam supports breakthrough research
A number of Abcam proteins and primary antibodies were used to demonstrate the potential of single cell western blot, including:
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Click on the image to expand.
(a) The scWestern array consists of thousands of microwells (20 μm in diameter, 30 μm deep) patterned in a 30-μm-thick photoactive polyacrylamide gel seated on a glass microscope slide. The array is comprised of 16 blocks of 14 × 30 microwells (6,720 in total) cast against an SU-8 photoresist master fabricated by soft lithography. E, electric field. Scale bar, 10 mm.
(b) Left, wide-field micrograph of a microwell block containing 15-μm fluorescent microspheres. Scale bar, 2 mm. Right, confocal micrograph of a live EGFP-expressing NSC settled in a rhodamine-tagged gel (GEL). Scale bar, 10 μm.
(c) Open-gel scWestern analysis is a 4-h, six-stage assay comprising cell settling, chemical lysis with a denaturing RIPA buffer, PAGE, UV-initiated protein immobilization onto the gel (hν, photon energy), diffusion-driven antibody probing (i.e., primary and fluorescently labeled secondary antibody probes: 1° Ab and 2° Ab*) and fluorescence imaging.
(d) PAGE resolves five fluorescently labeled proteins in a 550-μm separation distance (DRO, Dronpa, 27 kDa; OVA, ovalbumin, 45 kDa; BSA, bovine serum albumin, 66 kDa; OVA′, OVA dimer, 90 kDa; BSA′, BSA dimer, 132 kDa).
(e) scWestern analysis of EGFP and βTUB from a single NSC. RFU, relative fluorescence units. Distinct fluorescent dyes on each 2° Ab* enable multiplexed target analysis (EGFP, Alexa Fluor 488–labeled 2° Ab*; βTUB, Alexa Fluor 555–labeled 2° Ab*). Chemical stripping and reprobing allows multiplexed scWestern analysis.