affinity probes
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Separations ◽  
2022 ◽  
Vol 9 (1) ◽  
pp. 13
Author(s):  
Juli Novita Sari ◽  
Karthikeyan Kandasamy ◽  
Yu-Chie Chen

Escherichia coli are common pathogens, whereas E. coli O157:H7 is the most notorious E. coli strain, owing to its high virulence that can cause serious adverse effects and death. E. coli contains abundant peroxidases. Thus, the presence of E. coli can be determined by mixing E. coli with its substrate such as 3,5,3′,5′ tetramethylbenzidines (TMB) for endogenous peroxidase reactions. Under the presence of a high concentration of E. coli, colorless TMB turned to bluish, owing to the generation of the complexity of TMB and its oxidized TMB. To further reduce the detectable cell concentration, we developed an affinity-based method combined with an endogenous peroxidase reaction and mass spectrometric detection to detect E. coli. Affinity probes (diameter: ~20 µm) modified with maltose were generated for the enrichment of E. coli from sample solutions. E. coli trapped by the affinity probes was reacted with TMB in the presence of hydrogen peroxide for endogenous peroxidase reactions. Contactless atmospheric pressure ionization mass spectrometry was used for the detection of the reaction product, oxidized TMB (TMB cationic radical), to indicate the presence of target bacteria. The results showed that the developed method can be used to rapidly determine the presence of E. coli from a sample solution based on the detection of the TMB cationic radicals. The lowest detectable concentration of our method against E. coli O157:H7 in buffers and in complex juice samples was as low as ~100 cfu mL−1.


2021 ◽  
Author(s):  
Katherine T Grasso ◽  
Soumya Jyoti Singha Roy ◽  
Megan Jin Rae Yeo ◽  
Chintan Soni ◽  
Arianna O Osgood ◽  
...  

The E. coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNAEcTyr pair offers an attractive platform to genetically encode new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed for its engineering, has impeded its success in the past. Recently, we showed that EcTyrRS can be engineered using a facile E. coli based selection system, in a strain where the endogenous tyrosyl pair has been substituted with an archaeal counterpart. However, a significant cross-reactivity between the UAG-suppressing tRNACUAEcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNACUAEcTyr that overcomes this cross-reactivity. Optimized selection systems using this tRNA enabled efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide-dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporate useful ncAAs in mammalian cells, including photo-affinity probes, bioconjugation handles, and a non-hydrolyzable mimic of phosphotyrosine.


2021 ◽  
Author(s):  
Vladimir Khayenko ◽  
Clemens Schulte ◽  
Sara Lourenco Reis ◽  
Orly Avraham ◽  
Cataldo Schietroma ◽  
...  

We introduce Sylites - small and versatile fluorogenic affinity probes for high-contrast visualization of inhibitory synapses. Having stoichiometric labeling and exceptional selectivity for neuronal gephyrin, a hallmark protein of the inhibitory post-synapse, Sylites enable superior synapse staining compared with antibodies. Combined with super-resolution microscopy, Sylites allow precise nanoscopic measurements of the synapse. In brain tissue, Sylites reveal the three-dimensional distribution of inhibitory synapses within just an hour.


The Analyst ◽  
2021 ◽  
Author(s):  
Selda Glowacki ◽  
Maria Angela Gomes de Castro ◽  
Ka Man Yip ◽  
Ommolbanin Assadpour ◽  
Matthias Muenchhalfen ◽  
...  

We have developed a series of monovalent fluorophore-conjugated affinity probes based on the hapten 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP), which is widely used as a model antigen to study B lymphocytes and the...


2020 ◽  
Author(s):  
Andrew K. Rudd ◽  
Neel Mittal ◽  
Esther W. Lim ◽  
Christian M. Metallo ◽  
Neal K. Devaraj

ABSTRACTThe single-chained sphingolipid sphingosine is an essential structural lipid and signaling molecule. Abnormal sphingosine metabolism is observed in several diseases, including cancer, diabetes, and Alzheimer’s. Despite its biological importance, there are a lack of tools for detecting sphingosine in living cells. This is likely due to the broader challenge of developing highly selective and live-cell compatible affinity probes for hydrophobic lipid species. In this work, we have developed a small molecule fluorescent turn-on probe for labeling sphingosine in living cells. This probe utilizes a selective reaction between sphingosine and salicylaldehyde esters to fluorescently label sphingosine molecules. We demonstrate that this probe exhibits a dose-dependent response to sphingosine and is able to detect endogenous pools of sphingosine. Using our probe, we successfully detected sphingosine accumulation in live Niemann-Pick type C1 (NPC1) patient cells, a lipid transport disorder in which increased sphingosine mediates disease progression. This work provides a simple and accessible method for the detection of sphingosine and should facilitate study of this critical signaling lipid in biology and disease.


2019 ◽  
Vol 62 (21) ◽  
pp. 9521-9540
Author(s):  
Michael Chan ◽  
Fitzgerald S. Lao ◽  
Paul J. Chu ◽  
Jonathan Shpigelman ◽  
Shiyin Yao ◽  
...  

ACS Omega ◽  
2019 ◽  
Vol 4 (6) ◽  
pp. 9807-9812 ◽  
Author(s):  
Caroline A. Foley ◽  
Yazan A. Al-Issa ◽  
Kathryn P. Hiller ◽  
Seann P. Mulcahy

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