Single-cell western blotting

Hughes, Alex J.; Spelke, Dawn P.; Xu, Ze; Kang, Chi-Chih; Schaffer, David V.; Herr, Amy E.

doi:10.1038/nmeth.2992

Cited by 402 publications

(558 citation statements)

References 48 publications

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“…The bottom part of the figure illustrates the importance of follow-up functional experiments such as perturbation and high-throughput transcriptional assays 39,40 to validate inferred regulatory connections. 56 offer exciting perspectives, which will be enhanced further once protocols are developed that permit quantification of TF protein and target gene mRNA levels in the same single cell.…”

Section: Discussionmentioning

confidence: 99%

Regulatory network control of blood stem cells

Göttgens

2015

Blood

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Hematopoietic stem cells (HSCs) are characterized by their ability to execute a wide range of cell fate choices, including selfrenewal, quiescence, and differentiation into the many different mature blood lineages. Cell fate decision making in HSCs, as indeed in other cell types, is driven by the interplay of external stimuli and intracellular regulatory programs. Given the pivotal nature of HSC decision making for both normal and aberrant hematopoiesis, substantial research efforts have been invested over the last few decades into deciphering some of the underlying mechanisms. Central to the intracellular decision making processes are transcription factor proteins and their interactions within gene regulatory networks. More than 50 transcription factors have been shown to affect the functionality of HSCs. However, much remains to be learned about the way in which individual factors are connected within wider regulatory networks, and how the topology of HSC regulatory networks might affect HSC function. Nevertheless, important progress has been made in recent years, and new emerging technologies suggest that the pace of progress is likely to accelerate. This review will introduce key concepts, provide an integrated view of selected recent studies, and conclude with an outlook on possible future directions for this field. (Blood. 2015;125(17):2614-2620 Building blocks of transcriptional regulatory networksThe primary components of transcriptional regulatory networks are transcription factor (TF) proteins and the gene regulatory DNA sequences that they bind to.1 By binding to specific DNA sequence motifs within gene regulatory regions, TF proteins are central players for this primary step of decoding gene regulatory instructions. TF proteins typically contain a number of distinct modules, such as DNA binding, transcriptional activation, and protein/protein interaction domains, with the latter 2 being essential for the recruitment of the basal transcriptional machinery and the assembly of higherorder TF complexes. Based on sequence similarity within the DNA binding domain, TF proteins can be categorized into distinct families, such as homeobox, basic helix-loop-helix, or zinc finger TFs. Members of a given TF family often bind to similar DNA sequences, and proteinprotein interactions are common both within and between the different TF families.Individual TFs bind short sequence motifs that are often no longer than 4 to 6 bp. Any given 6-bp sequence will occur on average approximately once every 4000 bp. Consequently, there will be ;750 000 occurrences just by chance within the 3 000 000 000 bp of the human genome. The number of possible binding sites therefore far exceeds the 20 000 or so human genes, and it has long been assumed that only a small minority of all motif instances play a role in transcriptional regulation. Functional gene regulatory regions should therefore display characteristics that go beyond the mere occurrence of a specific sequence motif. One such characteristic is the presence of clusters of b...

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Section: Discussionmentioning

confidence: 99%

Regulatory network control of blood stem cells

Göttgens

2015

Blood

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“…To improve specific detection, the single cell western blot technique was developed to multiplex proteins within mammalian cells. 155 Rapid progress in proteomic profiling directs toward single cell proteomics.…”

Section: Single Cell Proteomicsmentioning

confidence: 99%

Cellular identity at the single-cell level

2016

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A single cell creates surprising heterogeneity in a multicellular organism. While every organismal cell shares almost an identical genome, molecular interactions in cells alter the use of DNA sequences to modulate the gene of interest for specialization of cellular functions. Each cell gains a unique identity through molecular coding across the DNA, RNA, and protein conversions. On the other hand, loss of cellular identity leads to critical diseases such as cancer. Most cell identity dissection studies are based on bulk molecular assays that mask differences in individual cells. To probe cell-to-cell variability in a population, we discuss single cell approaches to decode the genetic, epigenetic, transcriptional, and translational mechanisms for cell identity formation. In combination with molecular instructions, the physical principles behind cell identity determination are examined. Deciphering and reprogramming cellular types impact biology and medicine.

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“…single cell analysis. Recently, setting single cells into microwells, lysing in situ, separating, immobilizing and antibody probing are accomplished on a microscope slide [21]. These results suggest the potential of protein assays for rare cells collected by other methods.…”

Section: Western Blotting Using Microfluidic Chipsmentioning

confidence: 99%