Dual cross-linking ribonucleoprotein immunoprecipitation assay

2014 ◽  
Vol 92 (4) ◽  
pp. 317-319 ◽  
Author(s):  
Dilshad H. Khan ◽  
James R. Davie
Keyword(s):  
Uv Irradiation ◽  
Protein Interactions ◽  
Uv Light ◽  
Target Identification ◽  
Cross Linking ◽  
Immunoprecipitation Assay ◽  
Cross Linker ◽  
Subsequent Target ◽  
Protein Cross Linking

Ribonucleoprotein immunoprecipitation (RIP) is an antibody-based method to detect RNA–protein interactions in situ. In the assay, UV cross-linking is commonly used to preserve RNA–protein interactions for subsequent target identification. UV light is a zero-length cross linker and thus identifies proteins directly bound to RNAs. Here, we describe a dual cross-linking RIP method that involves sequential protein–protein cross-linking step with a protein–protein cross-linker, followed by protein–RNA fixation by UV irradiation. In this way, proteins that indirectly bound to RNA can be analyzed.

scholarly journals Differential Tandem Mass Spectrometry-Based Cross-Linker: A New Approach for High Confidence in Identifying Protein Cross-Linking

2016 ◽  
Vol 88 (20) ◽  
pp. 10215-10222 ◽  
Author(s):  
Jayanta K. Chakrabarty ◽  
Aishwarya G. Naik ◽  
Michael B. Fessler ◽  
Gerhard R. Munske ◽  
Saiful M. Chowdhury
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Cross Linking ◽  
Tandem Mass ◽  
High Confidence ◽  
New Approach ◽  
Cross Linker ◽  
Protein Cross Linking

scholarly journals A Fast Lysine Cross-linker DOPA Enables Mass Spectrometry Analyses of Protein Unfolding and Weak Protein-protein Interactions

2020 ◽  
Author(s):  
Jian-Hua Wang ◽  
Yu-Liang Tang ◽  
Rohit Jain ◽  
Fan Xiao ◽  
Zhou Gong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Guanidine Hydrochloride ◽  
Rnase A ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Protein Structure Analysis ◽  
Cross Linker

AbstractChemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) has become a widely used method for protein structure analysis. Central to this technology are chemical cross-linkers. The most popular cross-linkers are N-hydroxysuccinimide (NHS) esters, which react with protein amino groups relatively slowly over 10 minutes or more while in competition with the hydrolysis reaction of NHS esters. To improve the speed of cross-linking, we developed a new class of amine-selective and non-hydrolyzable di-ortho-phthalaldehyde (DOPA) cross-linkers. DOPA can cross-link proteins in 10 seconds under near physiological conditions, which is 60 times faster than the NHS ester cross-linker DSS. DOPA also works at low pH, low temperature, or in the presence of high concentrations of denaturants such as 8 M urea or 6 M guanidine hydrochloride. Further, DOPA-mediated pulse cross-linking captured the dynamic conformational changes associated with RNase A unfolding. Lastly, DOPA outperformed DSS at capturing weak but specific protein-protein interactions.

scholarly journals PVA and CS cross-linking combined with in situ chimeric SiO2 nanoparticle adhesion to enhance the hydrophilicity and antibacterial properties of PTFE flat membranes

RSC Advances ◽  
2019 ◽  
Vol 9 (33) ◽  
pp. 19205-19216 ◽  
Author(s):  
Chengcai Li ◽  
Hang Zhang ◽  
Feng Wang ◽  
Hailin Zhu ◽  
Yuhai Guo ◽  
...  
Keyword(s):  
Preparation Method ◽  
Low Cost ◽  
Antibacterial Properties ◽  
Cross Linking ◽  
Sio2 Nanoparticle ◽  
Simple Preparation ◽  
Cross Linker ◽  
Flat Membranes

Herein, a new hydrophilic and antibacterial PTFE flat MF membrane was fabricated via a low-cost and simple preparation method in which CS was crosslinked with PVA using ECH as a cross-linker followed by in situ chimeric SiO2 nanoparticle adhesion.

Aging Behavior of the Polyether Polyurethane Films Irradiated by UV

2013 ◽  
Vol 748 ◽  
pp. 16-21 ◽  
Author(s):  
Hai Yan Wang ◽  
Yu Wen Liu ◽  
Bin Sun ◽  
Shi Jie Huang ◽  
Ji Feng Tian
Keyword(s):  
Aromatic Ring ◽  
Uv Irradiation ◽  
Uv Light ◽  
Irradiation Time ◽  
Color Difference ◽  
Molecular Structures ◽  
Hydroxyl Group ◽  
Failure Behavior ◽  
Polyurethane Film

The failure behavior of polyether polyurethane films irradiated by UV depends on its molecular structures evolvement. The molecular structure evolvement of the polyether polyurethane films under UV irradiation were studied by in-situ FTIR spectra in this paper. It has been found that some oxygen contained groups such as hydroxyl group and carbonyl group increase; on the contrary, ether bond and CH2 decrease with the UV irradiation time. The aromatic ring maintains changeless. However, -NH group has not be determined due to the interference of the-OH and O-C=O. Results from FTIR and SEM have shown that UV photodegradative processes participated by oxidization occur mostly in ether segments and is accompanied by crosslinkage. Using the absorbance band of aromatic ring as the base the service life of polyether polyurethane can be determined by the change rate of C-O-C and-CH2 during UV irradiation. After UV irradiation, the polyether polyurethane film has higher color difference value and lower UV light transparence.

scholarly journals Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

2017 ◽  
Vol 114 (7) ◽  
pp. 1732-1737 ◽  
Author(s):  
Devin K. Schweppe ◽  
Juan D. Chavez ◽  
Chi Fung Lee ◽  
Arianne Caudal ◽  
Shane E. Kruse ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Mitochondrial Function ◽  
Mitochondrial Protein ◽  
Protein Structures ◽  
Protein Docking ◽  
Molecular Probes ◽  
Cross Linking ◽  
Chemical Cross Linking

Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I–III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.

In Situ Cross-linking Polymerization-Induced Self-Assembly: Not Only Generates Cross-linked Structures But Also Promotes Morphology Transition by the Cross-linker

2021 ◽  
Author(s):  
Jamshid Kadirkhanov ◽  
Cheng-Lin Yang ◽  
Zi-Xuan Chang ◽  
Ren-Man Zhu ◽  
Cai-Yuan Pan ◽  
...  
Keyword(s):  
Self Assembly ◽  
Cross Linking ◽  
Morphology Transition ◽  
Convenient Approach ◽  
Cross Linker ◽  
The Cross

Compared with the post-polymerization cross-linking strategy, in situ cross-linking by divinyl comonomers in polymerization-induced self-assembly (PISA) is a more straightforward and convenient approach to produce structurally stabilized nano-objects. However, cross-linking...

scholarly journals Photoelectrochemical Oxidation Assisted Catalyst-Referred Etching for SiC (0001) Surface

2021 ◽  
Vol 15 (1) ◽  
pp. 74-79
Author(s):  
Daisetsu Toh ◽  
Pho Van Bui ◽  
Kazuto Yamauchi ◽  
Yasuhisa Sano ◽  
◽  
...  
Keyword(s):  
Silicon Carbide ◽  
Uv Irradiation ◽  
Bias Voltage ◽  
Oxidation Rate ◽  
Uv Light ◽  
Removal Rate ◽  
Reaction Site ◽  
Photoelectrochemical Oxidation

In a previous study, we developed an abrasive-free polishing method named catalyst-referred etching (CARE) and used it for the planarization of silicon carbide (SiC) (0001). In this method, Si atoms at step edges are preferentially removed through a catalytically assisted hydrolysis reaction to obtain an atomically smooth and crystallographically well-ordered surface. However, the removal rate is low (< nm/h) and needs to be improved. In this study, we proposed an ultraviolet (UV) light assisted CARE method. In this method, UV light is irradiated onto a SiC surface to generate holes and oxidize the surface. The oxidized area, consisting of SiO2, can be quickly removed to form a nano-pit owing to the higher removal rate of SiO2 compared to that of SiC. The periphery of the nano-pits works as a reaction site, leading to a higher removal rate. To enhance the oxidation rate and form nano-pits, we applied electrochemical bias to the SiC substrate. However, the removal rate did not improve significantly when the bias voltage was higher than 3.0 V. This is because the electrochemical potential of Pt increased with the anodic potential of SiC, which oxidized the Pt surface and degraded the catalyst capability. To avoid this issue, we modified the catalytic pad, where an in-situ refreshment of the Pt surface is possible. As a result, the removal rate increased up to 200 nm/h at a bias of 7.0 V, which is 100 times higher than that of the CARE without UV irradiation. The proposed method is expected to contribute to the enhancement in the productivity and quality of next-generation SiC substrates.

Protein–protein and protein–DNA interactions in calf thymus nuclear matrix using cross-linking by ultraviolet irradiation

1986 ◽  
Vol 64 (5) ◽  
pp. 474-484 ◽  
Author(s):  
Teni Boulikas
Keyword(s):  
Uv Irradiation ◽  
Uv Light ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Micrococcal Nuclease ◽  
Cross Linking ◽  
Repeated Treatment ◽  
Major Protein ◽  
Protein Species ◽  
Calf Thymus

Nuclear matrices from calf thymus contained 30–50 protein species with one prominent band at 70 kilodaltons tentatively identified by its isoelectric point, apparent molecular weight, charge modification, and abundance as bovine lamin. The amount of DNA present in the matrix fraction was strongly dependent on the extent of digestion of the nuclei by micrococcal nuclease. The size of the DNA was higher than two kilobase pairs, although the chromatin DNA had been digested down to short oligonucleosomes. The lamin band was preferentially dissociated from isolated matrices during repeated treatment by 2 M NaCl or 5 M urea. Irradiation of calf thymus nuclear matrices at 313 nm induced protein–protein and protein–DNA cross-linking, as well as double-strand breaking of DNA, presumably at unprotected, protein-free regions. Lamin protein was more dramatically affected than other protein species by ultraviolet (UV) irradiation. In situ DNA hydrolysis, after the separation of the cross-linked matrix components on polyacrylamide – sodium dodecyl sulfate gels, followed by two-dimensional electrophoresis, showed lamin to be the major protein that was cross-linked to the DNA. Lamin molecules were also cross-linked by UV light to each other to form lamin homooligomers. A discrete size DNA fragment of approximately 450 base pairs is protected by lamin homooligomers from breakdown during UV irradiation. It is proposed that the direct contact between lamin and DNA found in this study is responsible for anchoring chromatin loops (domains) to a stable, immobile matrix structure.

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